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Stem Cells 2005;23:575-583 www.StemCells.com
© 2005 AlphaMed Press

Obese Diabetic Mouse Environment Differentially Affects Primitive and Monocytic Endothelial Cell Progenitors

Ola Awada, Chunhua Jiaob, Ning Mab, Martine Dunnwaldc, Gina C. Schattemanb

a Departments of Anatomy and Cell Biology,
b Exercise Science, and
c Dermatology, University of Iowa, Iowa City, Iowa, USA

Key Words. Angiogenesis • Endothelial cell • Diabetes • Monocyte • Progenitor cells • Somatic stem cells • Vascular development • Bone marrow cells

Correspondence: Gina Schatteman, Ph.D., Exercise Science 412 FH, University of Iowa, Iowa City, Iowa 52242, USA. Telephone: 319-335-9486; Fax: 319-335-6966; e-mail: gina-schatteman{at}uiowa.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Two classes of adult bone marrow–derived endothelial cell (EC) progenitors have been described, primitive hematopoietic stem cell–related cells and monocytic cells. Both differentiate into ECs and promote vascular growth in vivo but have distinct characteristics. Despite the association of obesity and type 2 diabetes with cardiovascular disease, their effects on primitive EC progenitors (prECPs) have not been examined, and the limited data on monocytic EC progenitors are conflicting. We investigated functional parameters of primitive and monocytic EC progenitors from obese diabetic (Leprdb) mice. The viability, proliferation, and differentiation of EC progenitors were unaffected in Leprdb cell cultures under basal condition. However, Leprdb-derived prECPs, but not monocytic EC progenitors, were less able to cope with hypoxia and oxidative stress, conditions likely present when EC progenitors are most needed. Intrinsic prECP dysfunction was also apparent in vivo. Whereas injection of nondiabetic prECPs promoted vascularization of skin wounds, Leprdb-derived progenitors inhibited it in nondiabetic mice. Additionally, although treatment with Leprdb-derived prECPs did not significantly reduce blood flow restoration to ischemic limbs, it resulted in increased tissue necrosis and autoamputation. Thus, type 2 diabetes coupled with obesity seems to induce intrinsic EC progenitor dysfunction that is exacerbated by stress. prECPs are more affected than monocytic progenitors, exhibiting a reduced ability to survive or proliferate. The proangiogenic phenotype of prECPs also seems to convert to an antiangiogenic phenotype in obese diabetic mice. These data suggest that therapies involving prECPs or stem-like cells in diabetic patients may be inadvisable at this time.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Diabetes is associated with a variety of cardiovascular disorders, including peripheral vascular disease and impaired neovascularization [1–5]. Therapies that increase vascularization tend to enhance wound healing, suggesting that treatments that improve neovascularization could have important clinical applications [1–5]. A subset of bone marrow–derived cells is believed to function as adult stem cells capable of differentiating into a variety of cell types, including endothelial cells (ECs) [6–9]. Endogenous bone marrow–derived cells play a poorly defined role in normal revascularization of injured tissues, but the ability of exogenous cells to promote vascular growth when administered as a local or systemic therapy is clear [10–13]. However, data are accumulating that the ability of bone marrow–derived cells to promote vascular growth is altered by diabetes, although exactly which bone marrow cells (BMCs) are impaired and the precise nature of the impairment are not known.

Studying putative EC progenitor dysfunction in diabetes is not simple, because the antigenic phenotype of bone marrow–derived cells capable of differentiating into ECs remains poorly defined, probably because it is plastic [14]. Nevertheless, studies on various subsets of blood and BMCs have provided ample evidence that there are at least two distinct classes of bone marrow–derived EC progenitors, primitive progenitors and monocytic-like progenitors. Primitive EC progenitors (prECPs) were the first identified, initially by expression of the hematopoietic stem cell antigens, CD34 and flk-1 [6], and subsequently by these and other hematopoietic stem cell antigens, notably CD133 (AC133) [15–17]. Later, several studies demonstrated that monocytes or monocyte-like cells can also function as EC progenitors, and it is these monocytic-like cells that are most commonly referred to as EPCs [18–21]. Further studies have shown that these two types of EC progenitors have distinct in vitro and in vivo properties [19, 22].

Both of these EC progenitor classes have been studied in the context of diabetes, but no distinction has been made between these two populations. Human CD34+ peripheral blood mononuclear cells are enriched for prECPs [6]. In culture, blood-derived CD34+ cells from type 1 diabetic but not type 2 diabetic subjects produced fewer ECs than those from nondiabetic controls [13]. Fewer ECs also were produced in cultures of adherent peripheral blood mononuclear cells, that is monocytic ECPs (mECPs), from type 1 and type 2 diabetic blood than nondiabetic blood [23, 24]. In addition, ECs derived from human type 2 diabetic mECPs exhibited reduced integration into vascular tubes in vitro [23].

In vivo, human nondiabetic blood-derived CD34+ cells promoted revascularization of skin wounds in type 1 diabetic mice [25]. In a nude mouse model of hind limb ischemia, exogenous nondiabetic blood-derived CD34+ cells had no effect on the restoration of blood flow to an ischemic limb in nondiabetic mice, but the same cells profoundly accelerated blood flow restoration in type 1 diabetic mice. Similarly, mouse BMCs enriched for murine hematopoietic stem cells dramatically improved vascularization of skin wounds in obese type 2 diabetic Leprdb but not congenic lean nondiabetic C57Bl/6 mice [26]. Moreover, when skin wounds of Leprdb mice were treated with Leprdb-derived hematopoietic stem cell–enriched BMCs, wound vascularization was severely inhibited [26]. In contrast, administration of whole BMCs from both nondiabetic and type 1 diabetic mice improved blood flow restoration in ischemic hind limbs of both nondiabetic and type 1 diabetic mice. However, the effect was greater in mice treated with nondiabetic than diabetic cells [27].

These studies indicate that the ability of bone marrow–derived cells to promote neovascularization as well as to differentiate into ECs may be impaired by diabetes. In type 1 diabetes, both functions may be compromised [13, 24], but the picture is less clear in type 2 diabetes, in which the data are conflicting and less complete. One explanation for some of the conflicting data is that the diabetes affects prECP and mECPs (i.e., adherent whole bone marrow after 4 days in culture) differently, but this has never been examined. Also, although the behavior of BMCs in diabetic and nondiabetic environments differs [13, 26], whether there is a negative synergism between the diabetic environment and diabetic BMCs has not been explored.

To study these issues, we developed a culture system for growth and differentiation of murine EC progenitors and investigated various functional properties of murine hematopoietic stem cells (i.e., mouse prECPs) and adherent BMCs (i.e., myeloid/monocytic EC progenitors) from Leprdb mice. We also compared the ability of nondiabetic and Leprdb prECPs to promote vascular growth in vivo in nondiabetic mice. Our data demonstrate that the obese type 2 diabetic syndrome induces intrinsic defects in prECPs but possibly not in mECPs. The defects in prECPs were evident in vitro by decreases in prECP-derived EC numbers after stress and in vivo in nondiabetic mice by their inhibition of vascular growth in skin wounds and exacerbation of ischemia-induced tissue damage in limb muscle.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
All mice were purchased from Jackson Laboratories (Bar Harbor, ME), and protocols were performed using procedures approved by the University of Iowa Animal Care and Use Committee.

Isolation and Culture of Mouse BMCs
C57Bl/6 or Leprdb male mice (8–10 weeks) were injected intraperitoneally with 150 mg/kg pentobarbital sodium. BMCs were collected from femurs and tibias and enriched for hematopoietic stem cell using Sca-1+ (Ly-6A/E+) magnetic bead–positive selection (Miltenyi, Auburn, CA) for in vitro studies and SpinSep lineage depletion (StemCell Technologies, Vancouver, BC, Canada) so that no beads were injected for in vivo studies, according to the manufacturer’s instructions. Hematopoietic stem cell–enriched cells will be referred to as prECPs hereafter. Enriched cells were R-PE rat anti-mouse Sca-1 (BD Pharmingen, San Diego) labeled and analyzed by fluorescence-activated cell sorting as described to assess purity [26]. Whole bone marrow was harvested from additional mice and preplated on tissue culture plastic and then placed in a tissue culture incubator for 1 hour to remove endothelial and stromal cell contaminants.

Freshly isolated diabetic and nondiabetic-derived prE-CPs and preplated BMCs from Leprdb and C57Bl/6 mice were plated in Medium D [19] with heat-inactivated fetal calf serum reduced from 20% to 7.5%. Cells were plated on 5 µg/cm2 pronectin-coated plastic (Deepwater, Woodward, OK) in 96-well trays at 1.2 x 105 cells per well for 3(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assays or in eight-well plastic chamber slides at 1.5 x 105 cells per well for immunolabeling at approximately 30% confluence. Some plates were placed in a hypoxia chamber (5% O2). Additional wells were stressed with 200 µM H2O2 at the time of plating or 1 and 4 days after plating. Medium was replaced at 4 days, at which time nonadherent cells were removed. The residual adherent mouse mECPs in the whole BMC cultures are analogous to human EPCs.

At 2 or 8 days, cell medium and any unattached cells were removed and relative cell numbers were determined by MTT assay according to the manufacturer’s instructions (ATCC, Manassas, VA). Briefly, cells were incubated with MTT reagent for 2 hours at room temperature and then in lysis reagent for 2 hours, and optical densities at 540 nm were measured. Assays were performed in duplicate or triplicate four to seven times for mECPs and in single wells or duplicate four to six times for prECPs. Cells in eight-well chamber slides were stimulated and stressed identically to cells in 96-well trays. At 8 days, cells were fixed with methanol and then immunolabeled with 2.5 µg/ml antivascular EC-cadherin (anti-VE-cadherin) (Cayman Chemical Co., Ann Arbor, MI) or isotype-matched immunoglobulin G (IgG) for 2 hours. Label was visualized by 1-hour incubation with 10 µg/ml Alexa 488 anti-rabbit IgG (Molecular Probes, Eugene, OR). Aortic smooth muscle cells served as negative and human umbilical vein ECs (HUVECs) as positive control cells.

To verify the EC phenotype of cultured cells, cells were fixed in methanol after 4 or 8 days in culture and labeled with Bandeiraea simplicifolia isolectin B4 (BSLB4) or immunolabeled with anti-tie 2 or anti-von Willebrand factor (vWF) as described [19] or with anti-VE-cadherin as above. Isotype-matched IgG and cell controls were performed as above.

Wounding
Male 8- to 10-week C57Bl/6J nondiabetic mice were anesthetized with isoflurane, and their back skin was depilated with Nair (Church & Dwight Co., Inc., Princeton, NJ). One day later, mice were again anesthetized, and two 6-mm bilateral full-thickness skin wounds were created on the dorsorostral back skin as described [26]. Three days later, mice were again anesthetized, and 2.5 x 105 freshly isolated prECPs in 25 µl 0.9% NaCl from either C57Bl/6 mice or type 2 diabetic male Leprdb (B6.Cg-m+/+Leprdb) mice [28] were injected under each wound. Controls received 25 µl 0.9 % NaCl. Forallmice, both wounds were injected with the same substance to avoid the possibility that injected cells could migrate or secrete substances into the contralateral wound.

Histology and Morphometry
Thirteen days after wounding, mice were anesthetized and depilated. The next day, they were lethally injected with sodium pentobarbital as above. Wound beds and underlying muscle surrounded by a margin of normal skin were harvested, fixed, and paraffin embedded as described [26]. Eight wounds (two from four different mice) in each group were serially sectioned (7 µm) perpendicular to the wound surface. Every 10th section throughout the entire wound bed was hematoxylin and eosin stained, and the adjacent section was immunolabeled with anti-CD31 (BD Pharmingen) to visualize blood vessels. Sections were treated for 3 minutes at 37°C with 100 µg/ml proteinase K (BD Pharmingen) before 1-hour incubation with 2.5 µg/ml anti-CD31 or rat IgG as control at 37°C in 0.75 µg/ml biotinylated anti-rat IgG and then 1:200 alkaline phosphatase-streptavidin complex (Vector, Burlingame, CA) followed by visualization with Vector Red (Vector) and hematoxylin counterstaining. The number of sections analyzed ranged from 15 to 20 per wound depending on the size of the wound.

Wound morphometry was performed as previously described [26]. Briefly, wound area was measured by digitally tracing the wound periphery (epidermis and dermis) of hematoxylin and eosin–stained sections. Wound volume was estimated by interpolation from the wound areas measured in every 10th section (i.e., every 70 µm) throughout the entire wound as described [25]. Similarly, the area of anti-CD31 immunolabeled blood vessels in the wound was measured digitally, and vascular volume was estimated by interpolation. The vessel volume density (vessel volume/wound volume) then was computed. Additionally, the vessel density (number of vessels per wound area) was computed. Finally, vessel size (vessel area per vessel number) was computed. Data were compared among groups using a one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference post-hoc analysis, and p < .05 was considered statistically significant [29].

Ischemic Limb
Left hind limbs of mice were depilated as above. One day later, surgical induction of unilateral hind limb ischemia was performed on 21 C57Bl/6 mice as described previously [13], and mice were divided into three groups. Two to 5 hours after surgery, mice were anesthetized with isoflurane and the medial thigh of the ischemic limb was injected intramuscularly with 5 x 105 freshly isolated prECPs from Leprdb (n = 6) or C57Bl/6 (n = 6) mice or vehicle (n = 9).

Blood Flow Analysis
Scanning LASER Doppler blood flow imaging (Moor Instruments Inc., Wilmington, DE) was used to assess blood flow restoration in mice after surgery as previously described [13, 30]. Blood flow was analyzed immediately before and after surgery and at 2, 4, 6, 8, and 11 days after surgery. Only mice whose mean flux in the operated limb immediately after surgery was ≤12% of that of the unoperated control limb were analyzed. Statistical comparisons of blood flow over time among groups were done by a repeated-measures ANOVA followed by Tukey’s honestly significant difference post-hoc analysis using SigmaStat software (SPSS Science, Chicago). p ≤ .05 was considered statistically significant. Data are presented as percent mean blood flux in the operated ischemic limb relative to flux in the unoperated control limb.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Culture of Murine EC Progenitors
Culture of mouse blood-derived mECPs has been limited, and these cells were only minimally characterized [31]. Culture of adult mouse bone marrow EC progenitors has not been reported, and we were unable to culture mECPs or prECPs in the conditions previously used for mouse peripheral blood mononuclear cells [31]. Hence, we first determined culture conditions in which murine mECPs and prECPs assume an EC phenotype. Cells were cultured on pronectin or fibronectin in Medium D [19]. Cells plated on fibronectin (which supports human mECPs) were not viable, whereas pronectin F (which does not support human mECP survival) supported growth of both mECPs and prECPs. The morphology of the mouse cells differed from that typically seen in human cultures. Cells remained round in the cultures for an extended period of time and never became truly spindle shaped, although some cells did elongate. Over time the cells began to cluster and eventually flattened into a more cobblestone-type morphology, particularly cells associated with the clusters (Fig. 1AGo).



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  		Figure 1. Phenotype of cultured mECPs. (A): Phase-contrast image of mECPs after 5 days in culture. Note the round morphology of cells and small clusters beginning to form. Inset shows a large flattened cell at 8 days. (B–F): Fluorescence micrographs of mECPs at 8 days in culture labeled with DAPI to visualize (B) nuclei, (C) anti-VE-cadherin, (D) control IgG (for VE-cadherin), (E) anti-tie-2, or (F) control IgG (for Tie-2). (B) and (C) are the same field. Note that essentially all cells in (C) are labeled with anti-VE-cadherin. (G, H): Phase-contrast images of mECPs at 8 days in culture labeled with (G) anti-vWF or (H) control IgG. Bar = 40 µm. Abbreviations: IgG, immunoglobulin G; mECP, mononuclear endothelial cell progenitor; VE-cadherin, vascular endothelial-cadherin; vWF, von Willebrand factor.

 
mECPs were immunolabeled at 4 or 8 days to determine whether they expressed the EC antigens VE-cadherin, tie-2, or vWF and tested for their ability to bind BSLB4. Data are summarized in Table 1Go. vWF expression was not detected in 4-day but was observed in 8-day cultures (Figs. 1G, 1HGo). BSLB4 binding and tie-2 immunolabeling were present at low levels in 4-day cultures. Both persisted at 8 days (Fig. 1EGo), but the levels did not seem to increase over this time period as tie-2 and Ulex lectin binding do in human cell cultures [18, 19]. (Human ECs do not bind BSLB4 but do bind Ulex lectin.) VE-cadherin expression also differed from that observed for human BMC-derived ECs. Whereas VE-cadherin was expressed only after relatively long-term culture of human BMCs [18, 19], 90% or more of cells in every 4-day culture examined (mean, 96%) labeled brightly with anti-VE-cadherin. This labeling persisted in 8-day cultures (Figs. 1B, 1CGo). No labeling was present in isotype-matched controls (Figs. 1D, 1F, 1HGo) or cultures of mouse hepatoma cells, whereas robust VE-cadherin and vWF expression was observed on HUVEC-positive controls (data not shown). VE-cadherin immunolabeling and BSLB4 lectin binding of prECP-derived cells also was performed at 4 and 8 days in culture, and both labels were detected at both times. Tie-2 immunolabeling was performed at 8 days, by which time prECPs were weakly labeled (data not shown).


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  		Table 1. Expression of endothelial cell antigens in endothelial cell progenitors over time
 
Diabetes and EC Progenitors in Basal Conditions
The ability of Leprdb and C57Bl/6 mECPs and prECPs to produce ECs was examined in culture. To begin, plating efficiency was compared. When equal numbers of freshly isolated cells were plated, the numbers of viable cells were similar in diabetic compared with nondiabetic mECP and prECP cultures 36–40 hours later (Fig. 2AGo). Cell numbers were then assessed 8 days after plating to assay cell growth (proliferation minus cell death).Cellnumbers did not differ significantly between nondiabetic and diabetic mECPs or prECPs at this time point (Fig. 2BGo). Because in humans it has been reported that diabetes leads to reduced proliferation of mECPs in culture at 7 days, this result was surprising. We considered the possibility that mouse mECPs survived in the diabetic mouse cultures but did not differentiate. Cells in 8-day cultures of mECPs were immunolabeled with VE-cadherin antibodies to identify ECs. Diabetes did not significantly affect the percentage of VE-cadherin–expressing cells; 95.8% ± 0.4% of nondiabetic and 97.2% ± 1.1% of diabetic cells were VE-cadherin positive.



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  		Figure 2. Effect of diabetes on cell numbers in ECP cultures. Freshly isolated mononuclear ECPs and prECPs from nondiabetic (ND) or Leprdb diabetic (D) mice were plated on pronectin in Medium D with 7.5% serum for (A) 2 days or (B) 8 days. Cell numbers were quantitated by 3(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide assay, and data are expressed as fold relative to the corresponding ND control. p > .05 as assessed by analysis of variance for data in both (A) and (B). Error bars = standard error of the mean. Abbreviation: prECP, primitive endothelial cell progenitor.

 
Diabetic mECPs Under Stress
BMCs seem to be mobilized in response to ischemia and used in repair of ischemic tissue. Hypoxia is a component of ischemia that could either stimulate mECP growth and differentiation or might exacerbate diabetes-induced mECP dysfunction. Thus, we examined the potential of nondiabetic and diabetic mECPs to produce EC in hypoxia. No significant effect of hypoxia on either nondiabetic or diabetic mECP numbers was observed at 2 days, indicating no effect of hypoxia on plating efficiency (Fig. 3AGo). Consistent with what we previously observed for nondiabetic human mECPs [22], culture in hypoxia also had no significant effect on nondiabetic and diabetic mouse mECP number in 8-day cultures (Fig. 3BGo).



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  		Figure 3. Effect of oxidative stress and hypoxia on cell numbers in mononuclear endothelial cell progenitor (mECP) cultures. Freshly isolated mECPs from nondiabetic (ND) or Leprdb diabetic (D) mice were plated on pronectin in Medium D with 7.5% serum. (A, B): mECPs were cultured in normoxia (norm) or 5% hypoxia (hyp) for (A) 2 days or (B) 8 days. (C, D): mECPs were not treated (cont) or treated with 200 µM H2O2 at (C) 1 day and assayed at 2 days or treated at (D) 1 and 4 days and assayed at 8 days. (E): mECPs were cultured in normoxia or hypoxia and treated at 1 and 4 days with 200 µM H2O2 and cultured for 8 days. Cell quantitation was done by 3(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide assay, and data are expressed as fold relative to normoxic ND controls. Error bars = standard error of the mean. Horizontal brackets indicate relevant pairs for which p < .05.

 
Diabetes leads to elevated levels of oxidative stress, and cells in ischemic tissues are subjected to increased levels of oxidative stress. Thus, the effect of elevated levels of oxidative stress via exposure to H2O2 on Leprdb and C57Bl/6 mECPs was examined. To induce oxidative stress, cells were treated with 200 µM H2O2 1 day after plating, and cell numbers were assessed at 2 and 8 days after plating. This is a high level of stress, but BMCs are resistant to oxidative stress–induced cell death [32]. We found that at H2O2 concentrations of 150 µM or less, no effects on EC progenitors were observed (data not shown).

H2O2 treatment reduced cell numbers in both Leprdb mECP (p < .05) and C57Bl/6 mECP (p < .01) cultures by 2 days, although the reduction in cell numbers was similar in the two groups (Fig. 3CGo). The number of cells remained lower in H2O2-treated cultures relative to controls at day 8, but the reduction in cell number was similar to that observed at day 2 (Figs. 3C, 3DGo). Because in humans mECPs have not begun to proliferate by 2 days in culture and the cells in the 2-day cultures were assayed only 12 hours after the addition of H2O2 in these experiments, the data suggest that H2O2 induces cell death but does not affect subsequent proliferation. No significant effect of H2O2 treatment on differentiation was apparent. The percentage of VE-cadherin–expressing cells was 95.8% ± 0.4% and 97.6% ± 1.2% in nondiabetic controls and H2O2-treated mECPs, respectively, and 97.2% ± 1.1% and 98.3% ± 1.0% in diabetic controls and H2O2-treated mECPs, respectively.

Because increased oxidative stress and hypoxia occur concomitantly in ischemic tissue, we also assessed the effect of the combination of the two on diabetic mECPs. The additional stress of hypoxia led to no further reduction in cell number in the dual treatment cultures compared with H2O2 alone (Fig. 3EGo).

Diabetic prECPs Under Stress
Because prECPs and mECPs have distinct properties, we also studied the effects of hypoxia and oxidative stress on prECPs. As with mECPs, hypoxia had no significant effect on prECP plating efficiency (Fig. 4AGo). In 8-day cultures, however, hypoxia stimulated nondiabetic prECPs (p < .01). Cell numbers in nondiabetic cell cultures increased by almost 50%, whereas hypoxia failed to significantly stimulate diabetic prECPs (Fig. 4BGo). Also, as with mECPs, H2O2 treatment resulted in decreased cell numbers in both Leprdb (p < .01) and C57Bl/6 (p < .05) prECP cultures (Fig. 4CGo). The reduction in cell numbers, however, was significantly greater in H2O2-treated diabetic than nondiabetic cultures, being reduced by one third relative to H2O2-treated nondiabetic controls (p < .01) (Fig. 4CGo).



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  		Figure 4. Effect of oxidative stress and hypoxia on cell numbers in primitive endothelial cell progenitor (prECP) cultures. Freshly isolated prECPs from nondiabetic (ND) or Leprdb diabetic (D) mice were plated on pronectin in Medium D with 7.5% serum. prECPs were cultured in normoxia (norm) or 5% hypoxia (hyp) for (A) 2 days or (B) 8 days. (C): prECPs were not treated (cont) or treated on day 1 with 200 µM H2O2 and cultured for 8 days. Cell quantitation was done by 3(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide assay, and data are expressed as fold relative to normoxic ND controls. Error bars = standard error of mean. Horizontal brackets indicate relevant pairs for which p < .05.

 
Diabetic prECP In Vivo Function
Because no differences in cultures of C57Bl/6 and Leprdb-derived mECPs in the in vitro studies were observed, we confined our in vivo studies to C57Bl/6 and Leprdb prECPs, which differed in their responses to stress in vitro. The in vivo studies were designed to test for intrinsic differences in the ability of diabetic Leprdb and nondiabetic C57Bl/6 prECPs to promote vascularization. Thus, the cells were tested in a nondiabetic environment.

Full-thickness skin wounds were created in nondiabetic C57Bl/6 mice. Cells or vehicle was injected under the wounds 3 days after wounding, and wounds were harvested 11 days later (14 days after wounding). In histological sections, marked differences between the three groups were apparent, with an increase in vascularization noted in nondiabetic cell treated and a decrease in Leprdb-cell treated wounds relative to controls (Figs. 5A–CGo). To quantitate these findings, the vascular volume density (vessel volume/wound volume) and vessel density (vessels per unit area) for cell- and vehicle-treated wounds were determined. In wounds treated with nondiabetic prECP, vascular volume density increased significantly (p < .05) (Fig. 5DGo), but vascular density was not significantly affected (Fig. 5EGo) relative to vehicle controls. Consistent with this, mean vascular size was increased in wounds treated with nondiabetic prECPs (p < .01) (Fig. 5FGo). In contrast, wounds treated with prECPs from Leprdb mice showed a dramatic decrease in both vascular volume density and vessel density (p < .01) (Figs. 5D, 5EGo) relative to controls, but the mean vessel size was not changed (Fig. 5FGo).



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  		Figure 5. Effects of primitive endothelial cell progenitors (prECPs) on skin wounds. Data from histological sections of mouse skin 14 days after creating full-thickness punch wounds and injecting with vehicle, nondiabetic (ND) prECPs, or diabetic (D) Leprdb prECPs (n = 7 to 8 for each group). (A–C): Bright-field micrographs of 7-µm sections labeled with anti-CD31 antibodies visualized with Vector Red (red) and stained with hematoxylin. Bar = 200 µm. (D–F): Mor-phometric analysis of vascularity. (D): Vascular volume density (vessel volume/wound volume) given as percent of the volume of wound tissue. (E): Vessel density (number of blood vessels per unit area of injured skin). (F): Mean vessel size. Error bars = standard error of the mean. Horizontal brackets indicate pairs for which p < .05.

 
We next tested whether the observed inhibition of vascularization was specific to skin wound healing. After femoral artery ligation, nondiabetic or diabetic prECPs or vehicle was injected intramuscularly into the ischemic limbs. The restoration of flow was followed by scanning LASER Doppler flow imaging (Fig. 6Go). Flow was improved by nondiabetic prECPs relative to limbs treated with Leprdb prECPs throughout the entire 11-day time period (p < .05). No significant effect of injection of Leprdb prECPs on flow restoration relative to vehicle-treated controls was observed, although there was a trend toward an inhibition of flow (Fig. 6Go). Moreover, injection of Leprdb-derived cells resulted in severe limb necrosis in five of six mice, whereas limb necrosis in control mice was much less severe, with only one mouse exhibiting severe necrosis.



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  		Figure 6. Blood flow restoration over time in ischemic hind limbs of nondiabetic mice as assessed by scanning laser Doppler analysis. Data are expressed as percent flux in ischemic limbs relative to contralateral control limbs. Limbs were injected with primitive endothelial cell progenitors (prECPs) from nondiabetic (n = 6) or Leprdb (n = 6) mice or vehicle (n = 7) on the day of femoral artery ligation to induce ischemia. Error bars = standard error of the mean. p < .05 for nondiabetic prECP versus diabetic prECP through day 11 and versus vehicle through day 8.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
The data presented here demonstrate that there are intrinsic differences between obese type 2 diabetic Leprdb and lean nondiabetic mouse–derived BMCs, but significant differences may be limited to or at least most profound for the most primitive (undifferentiated) cells. Bone marrow prECPs not only fail to produce normal numbers of ECs in conditions of stress but also inhibit vascular growth and exacerbate ischemia-induced tissue damage in nondiabetic mice.

We have yet to fully understand the nonhematopoietic functions of BMCs, but it is clear that there are at least two types that in some way promote neovascularization. prECPs, such as some Sca1+lin– BMCs, can function as hemangioblasts [33] and also promote vascular growth, possibly by secreting proangiogenic factors. Furthermore, because prECPs represent a large source of hematopoietic stem cells [34, 35], many if not all monocytic EC progenitors (i.e., mECPs) are likely derived from them. The less well-defined monocytic progenitors also differentiate into ECs in vivo and promote vascularization, also probably by secreting proangiogenic molecules [11, 19]. Interestingly, prECPs seem to modulate mECP function in vivo [19]. Thus, we considered it important to understand potential diabetes-induced changes in both primitive and monocytic EC progenitors.

Consistent with our findings for circulating CD34+ EC progenitors in humans [13, 19], type 2 diabetes does not seem to alter the ability of mouse prECPs or mECPs to produce EC under basal conditions in culture. However, because diabetes is associated with elevated levels of intracellular oxidative stress, the constitutive exposure to this stress might affect the ability of diabetic BMCs to withstand additional oxidative stress such as would be expected to increase during tissue injury. Our data indicate that H2O2-induced increases in oxidative stress leads to cell loss in both nondiabetic and Leprdb mECPs, but the effects are similar in the two groups. In contrast, Leprdb prECPs are more sensitive to H2O2-induced cell death than their non-diabetic counterparts. It remains to be determined why sensitivity of primitive but not monocytic EC progenitors increases. Perhaps they are equally sensitive but uptake or quenching of oxidants by other cells in the mECP cultures limits damage to the monocytic progenitors. Also, it is not known if sensitivity of the prECPs represents an increase in production or uptake of reactive oxygen species by the cells or decreased antioxidant enzyme production or activity, although preliminary data suggest the latter (G. Schatteman, unpublished data).

BMCs are active in tissue repair at times of tissue ischemia. Thus, if they are to differentiate into ECs, they need to be able to do so effectively in the context of hypoxia. We previously found that the ability of nondiabetic human mECPs to produce ECs in culture is unaffected by hypoxia [22]. However, the response of diabetic cells to hypoxia has not been examined. Our data show that as with nondiabetic human cells, hypoxia had no effect on type 2 diabetic or monocytic EC progenitors. In contrast, hypoxia greatly stimulated nondiabetic prECP growth, but Leprdb prECPs failed to respond to the stimulus.

Taken together, our in vitro data suggest that under basal conditions, EC progenitors can withstand the stress of obesity and diabetes. However, when coupled with additional stress, such as elevated oxidative stress, EC progenitor function is compromised. It seems that this sensitivity is confined to primitive cells, because even two additional stresses did not affect mECP function in obese diabetic mouse cells in our assays. Still, in vivo, when the mECPs would be subjected to multiple stresses simultaneously, the picture might be different. Furthermore, after longstanding hyperlipidemia and diabetes, mECP sensitivity might be increased. If the inability of diabetic BMCs to produce ECs in vitro reflects their in vivo functional abilities, a reduction in the integration of BMCs into the vascular endothelium could contribute to diabetes-associated impaired angiogenesis.

BMCs can promote vascular growth not only by integrating into the endothelium but also by secreting factors or in some way interacting with endogenous cells to create a more proangiogenic environment. Thus, diabetes has the potential to modulate this function of BMCs as well. Our earlier data demonstrated that, unlike their nondiabetic counterparts, prECPs from Leprdb mice can inhibit neovascularization in diabetic mouse wounds. This suggested intrinsic dysfunction in the Leprdb-derived cells but left open the question of whether an interaction between an unhealthy environment and dysfunctional cells was required for the inhibition. It was also unclear as to whether this inhibitory function was unique to skin wound healing or was true in other models of neovascularization.

Our finding that Leprdb prECPs inhibit revascularization of nondiabetic skin wounds clearly demonstrates that intrinsic changes in the obese diabetic mouse–derived prECPs are responsible for this inhibition. Moreover, the data suggest that these prECPs secrete factors that change the local milieu from proangiogenic to antiangiogenic. Because the number of vessels rather than the size of vessels is decreased, it is likely that it is the initial phase of vessel growth that is inhibited. Data from prECPs injected into ischemic limbs are consistent with, albeit less dramatic than, findings in skin wounds. Although there was no clear inhibition of neovascularization, there was a trend toward reduced flow in limbs treated with diabetic cells. We suspect that the same inhibitory effect is likely present in the ischemic limb but that the assay is less sensitive, perhaps because of volume dilution of the cells (i.e., the limb is much bigger than the skin wound). A more sensitive indicator of flow restoration may be limb functional improvement, and here we saw a marked deterioration in the limbs treated with Leprdb-derived cells relative to controls, because toe and foot autoamputation and limb necrosis were markedly increased.


   CONCLUSIONS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
Our results provide direct evidence for intrinsic defects in obese type 2 diabetic–derived murine prECPs. These defects are manifest as a reduced ability to produce ECs during stress and a change in their in vivo characteristics that renders them antiangiogenic rather than proangiogenic. It is disturbing that dysfunction is found in cells such as these that cycle relatively infrequently and are in the protected environment of the bone marrow. On the other hand, it is puzzling that effects of obesity and type 2 diabetes on their more differentiated progeny are not apparent. From a therapeutic standpoint, these data suggest that use of primitive stem or stem-like cells may not be advisable in diabetic patients at this time. Furthermore, until the dichotomy between primitive cells and their more differentiated progeny is better understood, even the use of mECPs should be approached with extreme caution.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
The authors wish to thank Raven Twitchell for assistance in preparation of this manuscript. This work was supported by grants from the Juvenile Diabetes Research Foundation (1-2001-534 to G.S.) and NIH (DK59223 and DK55965 to G.S.). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Juvenile Diabetes Research Foundation or NIH.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

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Received August 10, 2004; accepted for publication December 16, 2004.



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