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作者：Gina C. Schatteman, Ning Ma作者单位：Department of Exercise Science, University of Iowa, Iowa City, Iowa, USA
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1 U6 l. f: }/ i9 a# `* N1 d' E0 i 【摘要】
5 E& ?) U5 C+ l% c1 f3 O Local injection of hematopoietic stem cell¨Cenriched cells, including mouse lin¨C cells, accelerates vascularization in animal injury models, apparently by release of angiogenic factors. Locally injected lin¨C 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¨Cderived cells are currently being tested in clinical trials involving older patients, we investigated the effects of old lin¨C cells on skin wound vascularization in nondiabetic and obese diabetic mice. Treatment with old lin¨C 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¨Cderived cells may be poor candidates for therapeutic use in older patients and could actually harm them.
' h. O3 K+ p, y* K 【关键词】 Bone marrow Angiogenesis Aging Wound healing Sca- Endothelial progenitor cell) N: z5 |, _0 d9 m7 _& I8 _
INTRODUCTION5 | ?6 r& b Q. L+ U; w% p/ F
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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 . Whatever the mechanism, HSC-enriched populations hold promise in therapeutic settings in which accelerated vascularization would be advantageous.+ W9 m1 O" C! s: v
+ h+ Z0 \4 C% ?- o. m* T1 QAmong HSC-enriched populations that promote tissue vascularization are mouse bone marrow cells that express Sca-1 (Ly-6A/E) and lack lineage markers (lin¨C cells) and human blood cells that express CD34 . Thus, lin¨C or CD34 cells might be most helpful in environments in which angiogenesis is impaired, such as patients with diabetes or older patients.2 s' n. I' ^5 \/ Q( [+ K
0 s8 E0 o9 ~. gAn elevated risk for cardiovascular disease, endothelial cell dysfunction, and impaired angiogenesis is seen in aging and diabetic patients and animals . Thus, the ability of hematopoietic cells to promote tissue repair in general, and vascular growth in particular, may decrease with age.1 d& ], J% `) K1 L( j+ F8 [# D
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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 . Local treatment of skin wounds in Leprdb or congenic nondiabetic C57Bl/6 mice with Leprdb-derived lin¨C cells dramatically inhibits vascular growth, and ischemic limbs of C57Bl/6 mice treated with Leprdb lin¨C cells exhibit a higher rate of limb auto-amputation than do untreated or C57Bl/6 lin¨C-cell treated mice. That is, not only are lin¨C cells from obese diabetic mice not beneficial, they are actually harmful.
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+ e2 r4 ~2 Z3 X, n( B. ?To examine possible aging-related changes in the therapeutic potential of hematopoietic cells, we compared the effects of mouse lin¨C cells from young (2¨C4 month) and old (20¨C24 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.6 {; |# {, H7 B1 T9 Q2 e E
5 t; [' i) i, T/ ?MATERIALS AND METHODS7 L: d1 d8 U. h/ V( p* n9 }
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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).
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$ G1 y C. o, Y! P5 s$ @, ^% IWounding
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; l& e5 @8 O3 y7 B: w/ R$ m) `4 g; xMale (8¨C10 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%¨C1.1% 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 . Three days later, mice were again anesthetized, and 2.5 x 105 freshly isolated bone marrow cells enriched for mouse lin¨C in 25 µl 0.9% NaCl from either young (2¨C3 months) or old (20¨C24 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.
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Isolation of Mouse lin¨C Cells
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& t9 o4 D$ C# Y7 @Mice were lethally injected intraperitoneally with sodium pentobarbital (150 mg/kg). Bone marrow cells were collected from femurs and tibias, enriched for lin¨C 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 . Isolates ranged from 29% to 34% Sca-1 .1 B# A/ r( b) ?% R- a. E j
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Histological Procedures
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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.
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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¡ãC 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¡ãC 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.
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, d+ V/ S- L2 Z8 I. q% F" m) RMorphometry
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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., R$ j% \# V6 a1 ?1 X
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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 .
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$ G+ K: u8 e5 E! w9 E% Z1 IRESULTS: J i* p& k% {1 i$ S7 C$ K4 V) ]6 s
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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 .0 i' k( C0 e9 g9 C+ Z
# I! m* @( i9 a/ UFigure 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¨CF) C57B1/6 and (G¨CI) 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¨C cell¨Ctreated; and right column (F, I) shows old lin¨C cell¨Ctreated wounds that were treated 3 days after injury. Scale bar = 100 µm.1 b- c y+ V g& A
1 {. m7 ^8 `( y+ ?Young but Not Old lin¨C Cells Decrease Wound Volume9 S" [- N7 F f. b+ G* K( G
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Three days after skin wounding of C57Bl/6 and Leprdb mice (just prior to the start of revascularization ), young or old lin¨C 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¨C cells resulted in a nonsignificant trend (p . |4 E4 `* j% o8 u
# Y9 Y7 [/ L, Y/ aFigure 2. Skin vascularization. Brightfield micrographs of 7-µm histological sections of wounded skin that was treated with lin¨C 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¨C 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¨C cells and stained as in (A). Micrograph shows an unusually densely vascularized in a wound treated with old lin¨C 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).+ h; s* c! R, A3 N; b3 D& M
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Figure 3. Old lin¨C 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¨C2 mm3). (C): Percentage of vascular tissue in wound. Error bars = SEM. No cells = vehicle; Young = lin¨C cells from young C57Bl/6 mouse; Old = lin¨C cells from young C57Bl/6 mouse. Brackets indicate pairs for which p 0 f: h' W# i. j8 q
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Young lin¨C Cells Promote but Old lin¨C Cells Inhibit Wound Vascularization
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To assess the effect of lin¨C 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¨C cell¨Ctreated mice (Fig. 3B), but because young lin¨C 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 " A0 g: \' U! ^7 w
9 _0 y, j0 ~4 P1 tA change in vascular volume could be due to a change in either vessel size or vessel number. Treatment with young lin¨C 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¨C cells induced an increase in vessel cross-sectional area in C57Bl/6 but not in Leprdb mice (Fig. 4B) (p / k* m" f0 ~! a) Q1 Y+ {+ k
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Figure 4. Young and old lin¨C 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¨C2 µm2). Error bars = SEM. No cells = vehicle; Young = lin¨C cells from young C57Bl/6 mouse; Old = lin¨C cells from young C57Bl/6 mouse. Brackets indicate pairs for which p
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) ~% e4 b8 j6 V6 s* q/ S: z0 D0 {# cDISCUSSION
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* J+ o; |( H8 \- ` B9 v( \We examined the effects of murine lin¨C 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¨C 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¨C cells from old mice had no significant effect on wound size but profoundly impaired neovascularization in the wounds.
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Bone marrow cell injection alters both vessel number and size. There is a slight increase in vessel number density in young lin¨C cell¨Ctreated mice relative to vehicle-treated controls in Leprdb mice (Fig. 4A), whereas vessel number density decreases in mice treated with old lin¨C 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¨Ctreated mice, less than 56% in vehicle-treated mice, and less than 22% of vessels in young cell¨Ctreated 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¨C cells are observed in mice that heal normally, aging must induce intrinsic hematopoietic cell dysfunction.
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We previously found that despite inhibition of vascular growth by Leprdb-derived lin¨C 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¨Ctreated wounds. In contrast, old lin¨C cells failed to decrease wound size in Leprdb mice, suggesting that they might be more dysfunctional than those from young diabetic mice.- {) S, `. u0 u# }0 b& G2 y) m
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Changes in the inflammatory response could mediate the effects of lin¨C 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¨C cell¨Ctreated wounds . 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¨C cell¨Ctreated wounds is due to vasodilation.7 Z0 M8 o: S( D7 _
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CONCLUSION
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/ r( F3 `; t2 U1 wWe 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¨Cderived lin¨C cells, suggest that their antigrowth and maturation effects will be mediated through many of the same molecules.9 ` _: l5 z. E
) f) W# |9 ~5 \- e7 aAutologous 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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DISCLOSURES7 Y" G! P: Y2 J, K5 ]% p5 b
, O( W* v! F! z- pThe authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS8 y2 B& X; q- w" r$ l
' O( @# A6 M9 r5 h7 TThis 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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发表于 2009-3-5 00:09
发表于 2015-5-31 12:01
