Obese Diabetic Mouse Environment Differentially Affects Primitive and Monocytic Endothelial Cell Progenitors
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《干细胞学杂志》
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@uiowa.edu
ABSTRACT
Diabetes is associated with a variety of cardiovascular disorders, including peripheral vascular disease and impaired neovascularization . Therapies that increase vascularization tend to enhance wound healing, suggesting that treatments that improve neovascularization could have important clinical applications . 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) . 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 . 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 . 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 , and subsequently by these and other hematopoietic stem cell antigens, notably CD133 (AC133) . 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 . Further studies have shown that these two types of EC progenitors have distinct in vitro and in vivo properties .
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 . In culture, blood-derived CD34+ cells from type 1 diabetic but not type 2 diabetic subjects produced fewer ECs than those from nondiabetic controls . 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 . In addition, ECs derived from human type 2 diabetic mECPs exhibited reduced integration into vascular tubes in vitro .
In vivo, human nondiabetic blood-derived CD34+ cells promoted revascularization of skin wounds in type 1 diabetic mice . 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 . Moreover, when skin wounds of Leprdb mice were treated with Leprdb-derived hematopoietic stem cell–enriched BMCs, wound vascularization was severely inhibited . 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 .
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 , 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 , 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
Culture of Murine EC Progenitors
Culture of mouse blood-derived mECPs has been limited, and these cells were only minimally characterized . 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 . 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 . 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. 1A).
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 1. vWF expression was not detected in 4-day but was observed in 8-day cultures (Figs. 1G, 1H). BSLB4 binding and tie-2 immunolabeling were present at low levels in 4-day cultures. Both persisted at 8 days (Fig. 1E), but the levels did not seem to increase over this time period as tie-2 and Ulex lectin binding do in human cell cultures . (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 , 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, 1C). No labeling was present in isotype-matched controls (Figs. 1D, 1F, 1H) 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).
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. 2A). 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. 2B). 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.
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. 3A). Consistent with what we previously observed for nondiabetic human mECPs , culture in hypoxia also had no significant effect on nondiabetic and diabetic mouse mECP number in 8-day cultures (Fig. 3B).
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 . 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. 3C). 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, 3D). 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. 3E).
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. 4A). 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. 4B). Also, as with mECPs, H2O2 treatment resulted in decreased cell numbers in both Leprdb (p < .01) and C57Bl/6 (p < .05) prECP cultures (Fig. 4C). 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. 4C).
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–C). 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. 5D), but vascular density was not significantly affected (Fig. 5E) relative to vehicle controls. Consistent with this, mean vascular size was increased in wounds treated with nondiabetic prECPs (p < .01) (Fig. 5F). 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, 5E) relative to controls, but the mean vessel size was not changed (Fig. 5F).
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. 6). 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. 6). 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.
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
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
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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@uiowa.edu
ABSTRACT
Diabetes is associated with a variety of cardiovascular disorders, including peripheral vascular disease and impaired neovascularization . Therapies that increase vascularization tend to enhance wound healing, suggesting that treatments that improve neovascularization could have important clinical applications . 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) . 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 . 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 . 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 , and subsequently by these and other hematopoietic stem cell antigens, notably CD133 (AC133) . 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 . Further studies have shown that these two types of EC progenitors have distinct in vitro and in vivo properties .
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 . In culture, blood-derived CD34+ cells from type 1 diabetic but not type 2 diabetic subjects produced fewer ECs than those from nondiabetic controls . 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 . In addition, ECs derived from human type 2 diabetic mECPs exhibited reduced integration into vascular tubes in vitro .
In vivo, human nondiabetic blood-derived CD34+ cells promoted revascularization of skin wounds in type 1 diabetic mice . 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 . Moreover, when skin wounds of Leprdb mice were treated with Leprdb-derived hematopoietic stem cell–enriched BMCs, wound vascularization was severely inhibited . 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 .
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 , 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 , 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
Culture of Murine EC Progenitors
Culture of mouse blood-derived mECPs has been limited, and these cells were only minimally characterized . 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 . 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 . 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. 1A).
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 1. vWF expression was not detected in 4-day but was observed in 8-day cultures (Figs. 1G, 1H). BSLB4 binding and tie-2 immunolabeling were present at low levels in 4-day cultures. Both persisted at 8 days (Fig. 1E), but the levels did not seem to increase over this time period as tie-2 and Ulex lectin binding do in human cell cultures . (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 , 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, 1C). No labeling was present in isotype-matched controls (Figs. 1D, 1F, 1H) 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).
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. 2A). 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. 2B). 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.
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. 3A). Consistent with what we previously observed for nondiabetic human mECPs , culture in hypoxia also had no significant effect on nondiabetic and diabetic mouse mECP number in 8-day cultures (Fig. 3B).
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 . 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. 3C). 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, 3D). 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. 3E).
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. 4A). 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. 4B). Also, as with mECPs, H2O2 treatment resulted in decreased cell numbers in both Leprdb (p < .01) and C57Bl/6 (p < .05) prECP cultures (Fig. 4C). 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. 4C).
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–C). 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. 5D), but vascular density was not significantly affected (Fig. 5E) relative to vehicle controls. Consistent with this, mean vascular size was increased in wounds treated with nondiabetic prECPs (p < .01) (Fig. 5F). 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, 5E) relative to controls, but the mean vessel size was not changed (Fig. 5F).
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. 6). 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. 6). 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.
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
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.
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