First published online November 3, 2005
Stem Cells
Vol. 24 No.
3
March 2006, pp.
717
-721
doi:10.1634/stemcells.2005-0214; www.StemCells.com
© 2006 AlphaMed Press
TISSUE-SPECIFIC STEM CELLS
Old Bone Marrow Cells Inhibit Skin Wound Vascularization
Gina C. Schatteman,
Ning Ma
Department of Exercise Science, University of Iowa, Iowa City, Iowa, USA
Key Words. Bone marrow • Angiogenesis • Aging • Wound healing • Sca-1 • Endothelial progenitor cell
Correspondence: Gina C. Schatteman, Ph.D., Department of Exercise Science FH412, University of Iowa, Iowa City, Iowa 52242, USA. Telephone: 319-335-9486; Fax: 319-335-6966; e-mail: gina-schatteman{at}uiowa.edu
Received on May 12, 2005;
accepted for publication on October 28, 2005.
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ABSTRACT
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Local injection of hematopoietic stem cell–enriched cells, including mouse lin– cells, accelerates vascularization in animal injury models, apparently by release of angiogenic factors. Locally injected lin– cells from nondiabetic mice dramatically improve, but those from obese diabetic mice inhibit vascular growth in obese diabetic mouse skin wounds. Because of similarities between diabetes and aging and because autologous bone marrow–derived cells are currently being tested in clinical trials involving older patients, we investigated the effects of old lin– cells on skin wound vascularization in nondiabetic and obese diabetic mice. Treatment with old lin– bone marrow cells resulted in decreased vessel size and numerical density, leading to profoundly reduced vascular volume density in wounds of non-diabetic and diabetic mice. Our data suggest that bone marrow–derived cells may be poor candidates for therapeutic use in older patients and could actually harm them.
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INTRODUCTION
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A subset of hematopoietic stem or progenitor cells function as multipotent progenitors for nonhematopoietic cells. When bone marrow is reconstituted by single genetically tagged hematopoietic stem cells (HSCs) recruited from the bone marrow differentiate into a variety of cell types, including endothelial cells, in response to tissue injury [1–3]. Additionally, when HSC-enriched populations are administered locally, they can significantly improve healing and vascularization. However, few of these exogenous cells actually differentiate, and the cells are thought to mediate their therapeutic effects primarily through local cell– cell interactions or secretion of factors that promote tissue repair [4 –10]. Whatever the mechanism, HSC-enriched populations hold promise in therapeutic settings in which accelerated vascularization would be advantageous.
Among HSC-enriched populations that promote tissue vascularization are mouse bone marrow cells that express Sca-1 (Ly-6A/E) and lack lineage markers (lin– cells) and human blood cells that express CD34 [4–10]. These cells do not appear to induce vessel growth in nonischemic tissue, and even when injury is present, their effect on vascular growth depends on the environment. Thus, CD34+ and lin– cells do not improve, or only slightly improve, vascularization of ischemic limbs and skin wounds in nondiabetic mice [6, 8, 9, 11]. On the other hand, both CD34+ and lin– cells dramatically increase vascularization in injured diabetic mice [6, 8, 9, 11]. Thus, lin– or CD34+ cells might be most helpful in environments in which angiogenesis is impaired, such as patients with diabetes or older patients.
An elevated risk for cardiovascular disease, endothelial cell dysfunction, and impaired angiogenesis is seen in aging and diabetic patients and animals [12–17]. However, because HSCs can both serve as a source of endothelial cells and induce blood vessel growth, vascular disorders associated with these conditions might in part reflect dysfunction in hematopoietic cells. Certainly, aging-related changes in HSCs have been described [18 –20]. Furthermore, the ability of bone marrow–derived cells to produce functional endothelial cells appears to be reduced in both type 1 and type 2 diabetes [6, 8, 21–23]. Thus, the ability of hematopoietic cells to promote tissue repair in general, and vascular growth in particular, may decrease with age.
Because ideally one would use autologous cells for cell-based therapies and because the need for HSC-based therapy is likely to increase with age, it is important to understand the therapeutic potential of older hematopoietic cells. Furthermore, recent findings with cells from obese diabetic Leprdb mice make evaluation of old hematopoietic cells in this context more urgent. Leprdb mice lack functional leptin receptors, become obese shortly after birth, are insulin-resistant and hyperglycemic as adults, and exhibit impaired wound healing [24, 25]. Local treatment of skin wounds in Leprdb or congenic nondiabetic C57Bl/6 mice with Leprdb-derived lin– cells dramatically inhibits vascular growth, and ischemic limbs of C57Bl/6 mice treated with Leprdb lin– cells exhibit a higher rate of limb auto-amputation than do untreated or C57Bl/6 lin–-cell treated mice. That is, not only are lin– cells from obese diabetic mice not beneficial, they are actually harmful.
To examine possible aging-related changes in the therapeutic potential of hematopoietic cells, we compared the effects of mouse lin– cells from young (2–4 month) and old (20–24 month) mice on wound healing in normal-healing C57Bl/6 mice and healing-impaired Leprdb mice. Young cells had little effect on healing in C57Bl/6 mice but reduced wound size and increased vascularization of wounds in healing-impaired mice. In contrast, old cells did not affect wound size but profoundly inhibited vascular growth in normal and healing-impaired mice. Both vessel number and size were reduced. Our data suggest that autologous HSC-based cell therapy may not be appropriate in older patients.
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MATERIALS AND METHODS
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All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Iowa (Iowa City, IA).
Wounding
Male (8–10 weeks; The Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) nondiabetic C57Bl/6J and congenic obese diabetic Leprdb (B6.Cg-m+/+Leprdb) mice were anesthetized with 4% isoflurane at 2l per minute and then kept at 0.8%–1.1% at 1l per minute to maintain surgical anesthesia. Back skin was depilated with Nair (Church & Dwight Co., Inc., Princeton, NJ, http://www.churchdwight.com) and cleaned with povidone-iodine, and two full-thickness 6-mm punch skin wounds were created as described [8]. Three days later, mice were again anesthetized, and 2.5 x 105 freshly isolated bone marrow cells enriched for mouse lin– in 25 µl 0.9% NaCl from either young (2–3 months) or old (20–24 months) C57Bl/6 mice were injected in the dermis adjacent to each wound in a single injection. Controls received 25 µl 0.9% NaCl. For all mice, both wounds were injected with the same substance to avoid the possibility that injected cells could have systemic effects or could migrate or secrete substances into the contralateral wound. Eight wounds from four to six mice per group were analyzed.
Isolation of Mouse lin– Cells
Mice were lethally injected intraperitoneally with sodium pentobarbital (150 mg/kg). Bone marrow cells were collected from femurs and tibias, enriched for lin– cells using Spin Sep mouse hematopoietic progenitor negative selection kit (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), and assessed for Sca-1+ expression by fluorescence-activated cell sorting (FACS) using R-phycoerythrin rat anti-mouse Ly-6A/E (Sca-1) (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) as described [8]. Isolates ranged from 29% to 34% Sca-1+.
Histological Procedures
Mice were anesthetized and depilated 13 days after wounding and then lethally injected with sodium pentobarbital (150 mg/kg) the following day. Wound beds and underlying muscle surrounded by a margin of normal skin were harvested, fixed 4 hours in 100% methanol, processed through 100% ethanol and xylenes, and paraffin-embedded. Seven or eight wounds (one or two from four to six mice) in each group were serially sectioned (7 µm) perpendicular to the wound surface, rostral to caudally.
Every 10th section throughout the entire wound bed was stained with hematoxylin and eosin for wound analysis, and the adjacent section 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), incubated for 1 hour with 2.5 µg/ml anti-CD31 or rat immunoglobulin G (IgG) as a control at 37°C in 0.75 µg/ml biotinylated anti-rat IgG and then with 1:200 alkaline phosphatase-streptavidin complex (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), visualized with Vector Red (Vector Laboratories), and counterstained with hematoxylin and eosin. The number of sections analyzed ranged from 13 to 24 per wound, depending on the size of the wound.
Morphometry
All morphometric measurements were made by two blinded investigators. Data were similar for the two, and analyses of each investigator’s data set showed statistically significant differences between the same groups.
To determine wound area, the wound periphery (epidermis and dermis) of sections stained with hematoxylin and eosin was traced digitally from images (Nikon E600 microscope and DXM1200 camera, Nikon Corporation, Tokyo, http://www.nikon.com) using Metavue software (Universal Imaging Corporation, Downington, PA, http://www.universal-imaging.com) and analyzed as described [8]. Briefly, lateral wound boundaries were determined by the presence of intact hair follicles and organized epidermis and dermis as compared with few or no hair follicles, altered epidermal/dermal organization, and disorganization of collagen fibers within the wound. Wound volume was estimated by interpolation from the wound areas measured in every 10th section (i.e., every 70 µm) as described [9]. Similarly, vessel volume was estimated by interpolation from the area of anti-CD31 immunolabeled blood vessels in the wound measured every 70 µm. The percentage of vascular volume (vessel volume/wound volume) was calculated. The number of vessels per wound area was counted, and the mean cross-sectional vessel area (vessel area/vessel number) computed. Although vessels are expected to be randomly oriented in the wounds, all wounds were embedded and sectioned in the same orientation to avoid any potential misinterpretation of the data due to tissue anisotropy. Data were compared among groups using analysis of variance (ANOVA) with a Student-Newman-Keuls post hoc analysis, with p < .05 considered statistically significant [26].
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RESULTS
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Histological sections stained with hematoxylin and eosin demonstrate morphological differences in the skin of C57Bl/6 and Leprdb mice, as previously described [25, 27]. The skin of the nondiabetic C57Bl/6 mice is much thicker than that of healing-impaired obese diabetic Leprdb mice (Fig. 1A, 1B
). Therefore, the initial wound in Leprdb mice is typically half the size of that in the C57Bl/6 mice [8].
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Figure 1. Skin morphology and vascularization. Brightfield micrographs of histological sections of skin. 7-µm sections of skin from (A) C57Bl/6 and (B) Leprdb mice stained with hematoxylin and eosin. (C): Schematic of site of injection of vehicle or cells in the wound. 7-µm sections of (D–F) C57B1/6 and (G–I) Leprdb skin 14 days after wounding. Sections labeled with anti-CD31 antibodies visualized with Vector Red (red) and stained with hematoxylin. Left column (D, G) shows vehicle-treated; middle column (E, H) shows young lin– cell–treated; and right column (F, I) shows old lin– cell–treated wounds that were treated 3 days after injury. Scale bar = 100 µm.
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Young but Not Old lin– Cells Decrease Wound Volume
Three days after skin wounding of C57Bl/6 and Leprdb mice (just prior to the start of revascularization [28, 29]), young or old lin– cells or vehicle was injected under the wounds (Fig. 1C
). Wounds were harvested 11 days later (14 days after wounding) (Figs. 1D, 1F and 2). Treatment with either young or old lin– cells resulted in a nonsignificant trend (p < .09) toward decreased wound size in C57Bl/6 mice (Fig. 3A). In contrast, injection of young lin– cells resulted in a 42% reduction in wound volume (p < .03) in Leprdb mice, but no significant reduction was observed in the same mice treated with old lin– cells (Fig. 3A).
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Figure 2. Skin vascularization. Brightfield micrographs of 7-µm histological sections of wounded skin that was treated with lin– cells for 3 days and harvested 14 days after wounding. Sections stained with hematoxylin and labeled with anti-CD31 antibodies. (A): Skin of C57Bl/6 mouse treated with young lin– cells with anti-CD31 (bright red) labeled vessels throughout the wound. Note large arteriole-sized vessel (arrow). (B): High magnification image of skin of Leprdb mouse treated with old lin– cells and stained as in (A). Micrograph shows an unusually densely vascularized in a wound treated with old lin– cells. Note the many small vessels (arrowheads). Few anti-CD31 labeled cells that were not associated with lumens were found (arrow). Scale bar = 30 µm (A) and 15 µm (B).
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Figure 3. Old lin– cells inhibit vascular growth. Morphometric analysis of wound and vascular volume of 14-day skin wounds in nondiabetic C57Bl/6 and diabetic Leprdb mice. (A): Total wound volume (mm3). (B): Total vascular volume (x10–2 mm3). (C): Percentage of vascular tissue in wound. Error bars = SEM. No cells = vehicle; Young = lin– cells from young C57Bl/6 mouse; Old = lin– cells from young C57Bl/6 mouse. Brackets indicate pairs for which p < .05 (ANOVA with Student-Newman-Keuls post hoc analysis).
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Young lin– Cells Promote but Old lin– Cells Inhibit Wound Vascularization
To assess the effect of lin– cells on wound vascularization, the total vascular volume in the tissue was computed. Vascular volume in the wound was similar in vehicle and young lin– cell–treated mice (Fig. 3B), but because young lin– cells decrease wound size in Leprdb mice, the vascular volume density in the wounds of these mice was greater than that of vehicle controls (Fig. 3C) (p < .05). In contrast, both total vascular volume (Fig. 3B) and vascular volume density (Fig. 3C) were reduced significantly in all mice treated with old lin– cells (p < .01). As would be expected, in all wounds, vessels tended to be more dense near the wound periphery than in the center, especially near the bottom of the wound. This can be seen particularly clearly in Fig. 1E.
A change in vascular volume could be due to a change in either vessel size or vessel number. Treatment with young lin– cells had no significant effect on vessel numerical density (i.e., the number of vessels per cross-sectional area) in the wounds of C57Bl/6 mice, but density increased in Leprdb mice (Fig. 4A). Surprisingly, young lin– cells induced an increase in vessel cross-sectional area in C57Bl/6 but not in Leprdb mice (Fig. 4B) (p < .01). The fractions of vessels with cross-sectional diameters less than 10 µm were 55.9%, 4.7%, and 82.3% in mice treated with vehicle, young cells, and old cells, respectively. Vessel numerical density in old lin– cell–treated mice was only 68% and 62% of that of C57Bl/6 and Leprdb vehicle–treated mice, respectively (Fig. 4A) (p < .01). Even more dramatic were the old lin– cell–mediated changes in vessel cross-sectional area: The cells reduced mean vessel size by 74% in Leprdb mice (Figs. 2B, 4B) (p < .01). The fractions of vessels with cross-sectional diameters less than 10 µm were 42.0%, 21.2%, and 80.7% in obese diabetic mice treated with vehicle, young cells, and old cells, respectively. It was difficult to determine whether some of these small structures were in fact single cells. However, because staining was observed only occasionally when a lumen was not visible, the vast majority appeared to be capillaries (Fig. 2B).
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Figure 4. Young and old lin– cells have opposite effects on vessel size and density in wounds. Morphometric analysis of numerical density of blood vessels and mean size in 14-day skin wounds in nondiabetic C57Bl/6 and diabetic Leprdb mice. (A): Vessels per mm2 of wound. (B): Mean cross-sectional area (x10–2 µm2). Error bars = SEM. No cells = vehicle; Young = lin– cells from young C57Bl/6 mouse; Old = lin– cells from young C57Bl/6 mouse. Brackets indicate pairs for which p < .05 (analysis of variance [ANOVA] with Student-Newman-Keuls post hoc analysis).
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DISCUSSION
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We examined the effects of murine lin– cells on vascularization and healing of skin wounds in normally healing and healing-impaired mice. Our data demonstrate that bone marrow cells lose therapeutic potential with age, turning from pro- to anti-angiogenic agents. lin– cells from young mice greatly increase vascularization and decrease wound size in healing-impaired mice. In contrast, as with cells from obese diabetic mice, lin– cells from old mice had no significant effect on wound size but profoundly impaired neovascularization in the wounds.
Bone marrow cell injection alters both vessel number and size. There is a slight increase in vessel number density in young lin– cell–treated mice relative to vehicle-treated controls in Leprdb mice (Fig. 4A), whereas vessel number density decreases in mice treated with old lin– cells in both C57Bl/6 and Leprdb mice. Perhaps the more interesting difference was seen in effects on vessel size (Fig. 4B). Young cells induce an increase in vessel size, whereas old cells have the opposite effect in the wound. With a diameter cutoff of 10 µm, more than 80% in old cell–treated mice, less than 56% in vehicle-treated mice, and less than 22% of vessels in young cell–treated mice were capillaries or very small arterioles. Thus, whereas cells from young mice promote vessel maturation, those from old mice appear to inhibit vessel maturation. Because the inhibitory effects of old lin– cells are observed in mice that heal normally, aging must induce intrinsic hematopoietic cell dysfunction.
We previously found that despite inhibition of vascular growth by Leprdb-derived lin– cells in Leprdb mice, wound size was similar in Leprdb mice treated with Leprdb- or C57Bl/6-derived cells. That is, there was no coupling between vascularity and wound size (although collagen deposition and epidermal remodeling were slower) in the diabetic cell–treated wounds. In contrast, old lin– cells failed to decrease wound size in Leprdb mice, suggesting that they might be more dysfunctional than those from young diabetic mice.
Changes in the inflammatory response could mediate the effects of lin– cells. However, we observed no significant morphological differences in the inflammatory responses or the overall number of neutrophils and inflammatory cells 5 days after wounding (i.e., 2 days after cell injection) in vehicle and nondiabetic and diabetic lin– cell–treated wounds [11]. This, coupled with the fact that tissues were harvested 14 days after wounding (when inflammation is minimal), diminishes the likelihood that increased mean vessel diameter in young lin– cell–treated wounds is due to vasodilation.
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CONCLUSION
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We are currently attempting to identify molecular factors that mediate pro-angiogenic effects of HSCs derived from young nondiabetic mice and anti-angiogenic effects of HSCs derived from obese diabetic mice. Clearly, we should extend these studies to cells from old mice although physiological similarities between diabetes and aging, as well as the analogous behaviors of Leprdb and old mouse–derived lin– cells, suggest that their antigrowth and maturation effects will be mediated through many of the same molecules.
Autologous cell therapy could provide functional improvement in some settings, but our data suggest that such treatment might actually exacerbate vascular problems and poor healing in older patients. Still, because exogenous cells rarely incorporate into the vasculature, it might be possible to use allografts from young donors to achieve successful therapeutic outcomes in older patients. The allografts would need be present only long enough to induce vessel growth before being destroyed by the recipient’s immune system. Thus, in situations in which poor vascular growth sufficiently compromised a patient’s health, allografts might be attempted.
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DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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This study was made possible by National Institutes of Health grants DK59223 and AG10987. FACS was performed by the University of Iowa Flow Cytometry Facility.
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Abstract
Introduction
