- Email: [email protected]
db diabetic mice
VOLUME 69, NUMBER 4, AUGUST 2008
Effect of Cartilage Oligomeric Matrix Protein Angiopoietin-1 on Peripheral Nerves in db/db Diabetic Mice Heung Yong Jin, MD1; Ming Han Piao, MD1; Ji Hyun Park, MD1; Hong Sun Baek, MD1; Sik Lee, MD2; Won Kim, MD2; Sung Kwang Park, MD2; Chong Hwa Kim, MD3; Gou Young Koh, MD4; and Tae Sun Park, MD, PhD 1
1Endocrinology and Metabolism and Department of Internal Medicine, Research Institute of Clinical Medicine, Chon&k National UniversiU Medical School,ffeonju, South Korea; eRenal Regeneration Laboratory and Department of Internal Medicine, Research Institute of Clinical Medicine, Chonbuk National University Medical School,ffeonju, South Korea; 3Bucheon Sejong Hospital, Bucheon, South Korea; and 4Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea
ABSTRACT B A C K G R O U N D : Vascular and inflammatory processes have been reported to be factors
in the pathogenesis of diabetic neuropathy. Angiopoietin-1 (Angl) plays essential roles in regulating vascular growth, development, maturation, permeability, and inflammation. O B J E C T I V E : The aim of this study was to investigate the effect of cartilage oligomeric matrix protein (COMP)-Angl, which is a soluble, stable, potent Angl variant, on peripheral nerves in db/db diabetic mice. M E T H O D S : The db/db diabetic mice were randomized into 2 groups based on their weight and glucose level and treated with recombinant adenovirus (Ade), expressing either COMP-Angl or the ~-galactosidase gene (LacZ) (control), for 8 weeks. Immunohistochemistry was performed using a polyclonal antibody of antiprotein gene product and a secondary antibody. Intraepidermal nerve fiber density (IENFD) was quantified as nerve fiber abundance per unit length of epidermis (IENF/mm). In addition, the total capillary length (TCD per unit length of epidermis was summed (mm/mm2). All slides were coded and the capillary length and the number of nerve fibers were calculated by a blinded observer. R E S U L T S : Ten diabetic db/db mice (mean [SD] weight, 38.7 [1.95] g) were randomized to receive Ade-COMP-Angl or Ade-LacZ. IENFD was significantly greater in the Ade-COMP-Angl group compared with the Ade-LacZ group (mean [SD] 8.95 [3.30] vs 3.57 [0.73]/mm; P < 0.05). TCL was also significantly greater in the Ade-COMP-Angl group (2.79 [0.99] vs 2.04 [0.58] mm/mm2; P < 0.05). Compared with baseline, fasting blood glucose concentration after 8 weeks of treatment decreased significantly more in the Ade-COMP-Angl group than in the Ade-LacZ group (489 [45] to 361 [81] vs 495 [48] to 521 [70] mg/dL; P < 0.05). Accepted~r pub/ication March 27, 2008. © 2 0 0 8 Excerpta Medica Inc. All rights reserved.
doi:10.1016/j.curtheres.2008.08.002 0011-393X/$32.00
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CONCLUSIONS: These results suggest that Ade-COMP-Angl might have had proliferative effects on peripheral nerve and cutaneous capillaries in this small animal study. Further investigation of the metabolic effect, target site, and related mediator of COMP-Angl is needed. (Curr Ther Res C/in Exp, 2008;69:343 355) © 2008 Excerpta Medica Inc. K E Y W O R D S : COMP-angiopoietin-1, db/db diabetic mice, peripheral neuropathy.
INTRODUCTION
Despite the fact that peripheral neuropathy is a serious microvascular complication and a leading cause of foot amputation, which increases the mortality rate of diabetic patients, many study outcomes attempting to clarify pathogenic mechanisms or to successfully treat diabetic neuropathy have been unsatisfactory) Because hyperglycemia is a major factor in the development of diabetic neuropathy, strict control of blood glucose concentration is an essential strategy in the prevention and treatment of this condition. 2,3 Inflammation is increased in diabetes, and enhanced inflammatory response can cause nerve injury in experimental neuropathy. 4 In addition, neural cell degeneration and decreased nerve blood flow have been reported to be pathophysiologic features of diabetic neuropathy. 5 Treatment with growth factors and anti-inflammatory agents that prevent neural cell degeneration and improve blood flow directly or indirectly might be considered an effective therapeutic approach. In addition to promoting angiogenesis, angiopoietin-1 (Angl) is known to stabilize endothelium and to have anti-inflammatory effects6; however, it is unclear whether Angl also has a beneficial effect on peripheral nerve growth related to cutaneous vasculature in the diabetic animal model. Cartilage oligomeric matrix protein (COMP)-Angl is an Angl variant that is reported to be a more effective therapeutic agent than native Angl in the way of efficiency, generation, and potency.7,8 For systemic treatment with COMP-Angl, 2 adenoviral vectors were developed that encode the COMP-Angl gene and the ~-galactosidase gene (LacZ). By delivering adenovirus (Ade)-COMP-Angl using a viral vector, long-term, sustained COMP-Angl circulation can be maintained. 9 This study investigated the neuroprotective effect of Ade-COMP-Angl, a soluble, stable, potent Angl variant, by estimating intraepidermal nerve fiber density (IENFD) and cutaneous vascularity in db/db diabetic mice. MATERIALS COMP-ANG1
AND AND
METHODS
ADE-COMP-ANG1
PREPARATION
Ade-COMP-Angl was made from recombinant Chinese hamster ovary ceils. A recombinant adenovirus that could express COMP-Angl or LacZ was constructed using the pAdEasy vector system (Qbiogene, Inc., Irvine, California), as was done in previous studies .7,8 ANIMALS
Male rib/rib mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed in our pathogen-free animal facility; 8- to 10-week-old mice were used for this
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study. The animal care protocol and the experimental procedures were approved by the Institutional Animal Care and Use Committee of the Chonbuk National University Medical School (Jeonju, South Korea). STUDY
DESIGN
For this study, rib/rib mice that were similar in weight and blood glucose concentration were randomized based on weight and glucose level into 2 groups, and 109 plaque-forming units of Ade-COMP-Angl or Ade-LacZ diluted in 50 laL of sterile 0.9% sodium chloride was injected intravenously through the tail vein for adenoviral treatment. Serum concentrations of Ade-COMP-Angl remained at elevated concentrations for 2 weeks, as previously described. 1° Consequently, Ade-COMP-Angl or AdeLacZ was injected 4 times at 2-week intervals. In a preliminary study, we found that LacZ had no effect on weight, blood glucose concentration, or other measured parameters in the same animal model. Therefore, LacZ was used as the control agent. Weight, blood glucose concentration, and food intake were measured at baseline and at 2-week intervals. Blood glucose concentration was measured in a sample drawn from a tail vein using Precision Xtra Plus ® (Abbott Laboratories, MediSense Products, Bedford, Massachusetts), and serum insulin concentration was analyzed using a radioimmunoassay (Neodine Laboratories, Seoul, South Korea). After 8 weeks of treatment, the mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and a punch biopsy of skin 3 m m in diameter was performed on the dorsum of both feet to quantify the degree of innervation and vascularity. IMMUNOHISTOCHEMISTRY
For immunohistochemistry on frozen microtome sections, skin tissue specimens were fixed with periodate-lysine-paraformaldehyde (2 % paraformaldehyde, 0.075 M lysine, 0.05 M phosphate buffer p H 7.4, 0.01 M sodium m-periodate) solution for 24 hours. After thorough rinsing in phosphate-buffered saline (PBS), which contained 20% glycerol and 0.1 M phosphate buffer, for 48 hours at 4°C, tissue specimens underwent cryoprotection with Tissue-Tec ® (OCT compound) (Miles, Elkhart, Indiana). Sections 40 lam were cut on a sliding microtome (Leica CM 1510 ®, Leica Microsystems AG, Wetzlar, Germany) perpendicular to the dermis and were immerged in PBS for 15 minutes at room temperature. Samples were then transferred into microtubules containing Dako Protein Block Serum-Free ® (Dako Corporation, Carpinteria, California) as a blocking buffer supplemented with 3% goat serum. After 30 minutes in the blocking buffer on a shaker table at room temperature, sectioned specimens were washed with PBS twice for 10 minutes and then incubated separately overnight at 4°C in 1 of 2 primary antibodies rabbit antiprotein-gene-product 9.5 (PGP 9.5) (Biogenesis, Poole, United Kingdom) for peripheral nerves at a dilution of 1:100 or hamster antimouse CD31 (1:100) (Chemicon International Inc., Temecula, California) for capillary endothelial ceils. The antibodies were diluted in antibody diluent (Dako Corporation) supplemented with 1% goat serum. After a sterile washing, the relevant secondary a n t i b o d i e s ~ o a t antirabbit immunoglobulin G (IgG)-FITC (1:200) or Cy3 Aff]niPure goat antiarmenian hamster IgG (heavy and light chain) (1:200) (Jackson
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ImmunoResearch Laboratories Inc., West Grove, Pennsylvania) were separately loaded (ie, immunostained) for 1 hour at room temperature in a dark room. After washing with PBS as above, sections were placed on slides and prepared with fluorescent mounting medium (Dako Corporation). QUANTIFICATION
OF INNERVATION
AND
CAPILLARY
VASCULARITY
PGP 9.5 immunoreactive nerve fibers in the epidermis of each section were counted at a magnification of 100x. Each individual nerve fiber with branching points inside the epidermis was counted as 1 fiber. For epidermal nerve fibers with branching points inside the dermis, each individual nerve fiber was counted separately. Conventional IENFD was derived and expressed as epidermal nerve fiber abundance per unit length of the epidermis (IENFD/mm). IENFD was calculated by a blinded observer, and all biopsy slides were coded and mixed with a set of normal slides to ensure examiner blinding. Photomicrographs of representative sections were captured using a digital camera (Axiocam HRC ®, Carl Zeiss, Goettingen, Germany). Capillary vascularity was also observed (by a blinded investigator) using the same method that was used to determine IENFD, including the photomicrographs. Images were collected and analyzed using a microscope (Axioskop2, Carl Zeiss Ltd., Welwyn City, United Kingdom) and the Axiovision 5.1 program (Carl Zeiss Ltd.). STATISTICAL
ANALYSIS
A power analysis (90%) was performed to determine sample size. Based on the power analysis, 5 mice per group were needed to detect significant between-group differences. Data were presented as mean (SD). The unpaired t test was used to compare the AdeLacZ and Ade-COMP-Angl groups. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 12.0 software (SPSS Inc., Chicago, Illinois). RESULTS ANIMALS
Five mice (mean [SD] weight, 38.7 [1.95] g) were assigned to each of the 2 groups. EFFECTS
OF ADE-COMP-ANG1
ON CHARACTERISTICS
OF
dbldb M I C E
Characteristics of db/db mice after IV administration of Ade-LacZ or Ade-COMPAngl for 8 weeks (4 injections at 2-week intervals) are presented in Table I. Before treatment (baseline), the mice in the 2 groups did not differ significantly in age (data not shown), weight, food intake, or fasting blood glucose concentration. Although there was no significant difference in food intake between the groups during the experimental period, both body weight (51.8 [1.7] vs 54.1 [2.2] g; P < 0.05) and fasting blood glucose concentration (361 [81] vs 521 [70] mg/dL; P < 0.05) were significantly lower in the Ade-COMP-Angl group compared with the Ade-LacZ group. However, there was no significant between-group difference in serum insulin concentration after treatment (39.5 [5.1] vs 39.7 [3.9] laU/mL) (Table I).
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H.Y. JIN ET AL.
Table I. Characteristics of the diabetic db/db mice at baseline and after 8 w e e k s of treatment (N = 1 0 ) . Data are mean (SD).
Ade-COM P-Angl (n = 5) Characteristic Weight, g Food intake, g / 2 4 h
Ade-LacZ (n = 5)
Baseline
Week 8
Baseline
38.6 (2.1)
51.8 (1.7)*
38.8 (1.9)
54.1 (2.2)
7.8 (0.8)
8.2 (0.7)
7.8 (1.1)
Serum insulin, pU/mL
8.3 (0.5) 39.5 (5.1)
Fasting blood glucose, mg/dL
4 8 9 (45)
Week 8
39.7 (3.9)
361 (81)*
495 (48)
521 (70)
IENFD, no./mm
8.95 (3.30)*
3.57 (0.73)
Total capillary length per unit length of epidermis, mm/mm 2
2.79 (0.99)*
2.04 (0.58)
Ade = adenovirus; COMP = cartilage oligomeric matrix protein; Angl = angiopoietin-1; LacZ : ~-galactosidase gene; IENFD= intraepidermal nerve fiber density. *P < 0.05 versus Ade-LacZ-treated group. E F F E C T S OF A D E - C O M P - A N G 1
ON I N T R A E P I D E R M A L
NERVE FIBERS
Mean (SD) IENFD was significantly greater (>2-fold) in the Ade-COMP-Angl group compared with the Ade-LacZ group (8.95 [3.30] vs 3.57 [0.73]/mm; P < 0.05). IENFD of each mouse is shown in Table II. The morphologic findings o f l E N F D in each group appeared to increase (Figure 1). E F F E C T S OF A D E - C O M P - A N G 1
ON T O T A L C A P I L L A R Y
LENGTH
Mean (SD) total capillary length (TCL) was also significantly greater in the AdeCOMP-Angl group than in the Ade-LacZ group (2.79 [0.99] vs 2.04 [058] ram/ram2; P < 0.05). TCL for each mouse is shown in Table II; the increased pattern ofvascularity Table II. Intraepidermal nerve fiber density (IENFD) and total capillary length (TCL) of the individual diabetic db/db mice after 8 w e e k s of t r e a t m e n t with adenovirus (Ade)-cartilage oligomeric matrix protein-angiopoietin-1 ( C O M P - A n g l ) or A d e - ~ - g a l a c t o s i d a s e gene (LacZ). Data are mean (SD).
Ade-COM P-Angl-Treated IENFD Mouse Mouse Mouse Mouse Mouse
1 2 3 4 5
Mean (SD)
6.18 12.52 8.98 7.32 9.76
(1.23) (6.37) (3.71) (2.36) (4.94)
8.95 (3.30)
TCL 3.33 2.72 2.88 2.18 2.85
(0.72) (0.57) (0.71) (0.38) (2.61)
2.79 (0.99)
Ade-LacZ-Treated IENFD 3.22 4.88 3.32 3.17 3.29
(0.74) (1.74) (2.17) (0.63) (1.22)
3.57 (0.73)*
TCL 2.36 1.83 2.07 1.95 2.01
(0.59) (0.62) (0.54) (0.56) (0.61)
2.04 (0.58)
*P < 0.05 versus Ade-COMP-Angl-treated group.
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CURRENT THERAPEUTIC RESEARCH
Figure 1. Intraepidermal nerve fiber density (IENFD) in db/db diabetic mice after 8 weeks of treatment with (A) adenovirus (Ade)-carUlage oligomeric matrix protein-angiopoietin-1 (COMP-Angl) or (B) Ade-~-galactosidase gene (LacZ). Arrows indicate the peripheral nerves maintained in the epidermis, which was branched in the dermis (200X); capillary endothelial immunostaining with hamster antimouse CD31. (continued)
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in the Ade-COMP-Angl group is presented in schematic fashion in Figure 2 (including photomicrographs). DISCUSSION
Diabetic neuropathy is one of the most important factors affecting the prognosis of diabetic patients. Approximately half of diabetic patients are reported to have neuropathic symptoms. 11 In particular, significant morbidity among diabetic patients is associated with sensory deficit of the lower extremities, potentially resulting in foot ulceration or amputation. Prevention and treatment of diabetic neuropathy have often been unsatisfactory. The effects of various agents on a hypothesized pathogenic cause of
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Figure 2. Cutaneous capillary vascularity shown in schematic representations of skeletonized microvessels of db/db diabetic mice after 8 weeks of treatment with (A) adenovrius (Ade)-cartilage oligomeric matrix protein-angiopoieUn-1 (COMP-Angl) and (B) Ade-13-galactosidase gene (LacZ) (200X; capillary endothelial immunostaining with hamster antimouse CD31). (continued)
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diabetic neuropathy have been investigated in both experimental and clinical trials. Most of these agents have been found to have promising results in experimental studies, but the findings in clinical trials have been less convincing. 12,13 Several factors, including hyperglycemia, the polyol pathway, protein kinase C (PKC), and nonenzymatic glycation have been postulated to be involved in the pathogenesis of diabetic neuropathy. 14 16 Most therapeutic agents for the treatment of diabetic neuropathy have been focused on these putative mechanisms. 17 21 Reports suggest that vascular factors, including microangiopathy or endoneural ischemia, and neuropathic processes may be associated with diabetic neuropathy. 2°,22 In addition, studies have found that increased inflammation in diabetes can cause peripheral nerve injury.4 New anti-inflammatory strategies targeting both the neurotrophic and angiogenic aspects of diabetic neuropathy may be possible therapies. Angl is a ligand of the Tie2 receptor, which is a member of the tyrosine kinase family that is expressed on vascular endothelial ceils. Angl, which is mediated by phosphorylating the Tie2 receptor and signaling via the Akt pathway, has therapeutic potential in angiogenesis and vascular endothelial dysfunction; however, Angl has aggregation and insolubility problems due to the superclustering domain and multimerization of Angl. 23 For these reasons, a chimeric form of Angl with COMP (COMP-Angl) was developed to avoid these limitations. COMP-Angl is a soluble, stable, potent Angl variant that has been found to be effective in preventing endothelial dysfunction and inflammation.7,8 However, the effect of COMP-Angl on peripheral neuropathy in diabetes has not yet been studied in experimental models. Therefore, we investigated the effects of COMP-Angl on peripheral nerves of,rib/rib diabetic mice. Inflammatory mediators have been reported to be pathogenic mechanisms of diabetic neuropathy. 24,25 We reported that the anti-inflammatory effect of COMPAngl involves inhibiting the nuclear factor (NF)-KB pathway, which subsequently decreases activation of transforming growth factor (TGF)-~. 1° We also reported that COMP-Angl could increase blood flow by improving endothelial survival and vascular size. 26'27 Vascular factors related to tissue blood flow that can be targeted for treatment in diabetic peripheral neuropathy include increasing endothelium-derived relaxing factor, inhibition of PKC, suppression of NF-KB, and modulation of vascular endothelial growth factor (VEGF) subtype. Several factors that have an angiogenic effect (eg, VEGF, hepatocyte growth factor, fibroblast growth factor) have been reported to have therapeutic potential in diabetic neuropathy. 28 30 Neurotrophic factors, such as nerve growth factor, have been studied in animal models of diabetic neuropathyS; however, the clinical usefulness of this therapy was limited by the absence of an established therapeutic dose and reliable data. In this study, the effect of COMP-Angl might not have been mediated by direct action on peripheral nerves; rather, COMP-Angl may have triggered the activation of a cytokine cascade involved in nerve regeneration or inhibition of nerve degeneration in association with angiogenesis. Although previous studies have identified the effect of COMP-Angl on cytokines, a variety of cytokines will need to be investigated (including histologic analysis) to clarify the mechanism of action of COMP-Angl in angiogenesis and nerve regeneration in diabetic neuropathy. 8 10
352
H.Y. JIN ET AL.
In this study, fasting blood glucose concentration was significantly lower in db/db mice treated with Ade-COMP-Angl compared with those treated with Ade-LacZ. However, the serum insulin concentration was not significantly difterent between the 2 groups. These results suggest that the insulin-sensitizing eftect and resulting metabolic eftects of COMP-Angl might also contribute to neuroprotection in diabetic mice. Therefore, the metabolic eftect of COMP-Angl in diabetic mice, including its exact mechanism of action, and another group showing similar glucose control with COMPAngl warrant further investigation. In our study, IENFD and TCL were greater in the group treated with Ade-COMPAngl compared with the Ade-LacZ group. An increased number of nerve fibers and enriched vascularity were observed in the series of photomicrographs in the AdeCOMP-Angl group. LIMITATIONS
Our study had several limitations. The effects of COMP-Angl on peripheral nerves and capillaries might be related to its glucose-lowering effects. Next, if IENFD and TCL in both groups were measured prior to treatment and compared with our findings after treatment, it would have been more evident whether COMP-Angl indeed had a proliferative effect on diabetic peripheral neuropathy. Therefore, we cannot definitively conclude that COMP-Angl protected the mice from any subtle loss of nerve fibers or vascularity or that it was associated with improvement in these factors when compared with LacZ. Finally, the presence of COMP-Angl receptors in the peripheral nerves and the related mediators such as TGF-~ or NF-KB, were not investigated in this study (except to observe the morphologic changes), which would reveal more precise evidence on COMP-Angl effects. CONCLUSIONS
These db/db diabetic mice treated with Ade-COMP-Angl had greater IENFD and cutaneous capillary length compared with the Ade-LacZ group in this small animal study. We suggest that Ade-COMP-Angl might have therapeutic potential, with a dual impact on peripheral nerves and cutaneous capillaries in this diabetic animal model. Ade-COMP-Angl treatment might also improve blood glucose concentration. Further studies targeting molecular markers and COMP-Angl receptors in peripheral nerves are warranted to determine the direct eftect of COMP-Angl on neural ceils. ACKNOWLEDGMENTS
This work was supported in part by grants from the National Research Laboratory Program of Korea Science and Engineering Foundation (Jeonju, South Korea) and the Medical Science Education Program for the 21st Century of Chonbuk National University Medical School. REFERENCES
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RESEARCH
2. The Diabetes Control and Complications Trial Research Group. The effect of intensive diabetes therapy on the development and progression of neuropathy. Ann Intern Ned. 1995;122:561 568. 3. Test;ayeS, Stevens LK, Stephenson JM, et al. Prevalence of diabetic peripheral neuropathy and its relation to glycaemic control and potential risk fiactors: The EURODIAB IDDM Complications Study. Di~betologi~. 1996;39:1377 1384. 4. Wang Y, Schmeichel AM, Iida H, et al. Enhanced inflammatory response via activation of NF-kappaB in acute experimental diabetic neuropathy subjected to ischemia-reperfusion injury. J Neurol Sci. 2006;247:4~52. 5. Goss JR, Goins WF, Lacomis D, et al. Herpes simplex-mediated gene transfer of nerve growth fiactor protects against peripheral neuropathy in streptozotocin-induced diabetes in the mouse. Diabetes. 2002;51:2227 2232. 6. Suri C, McClain J, Thurston G, et al. Increased vascularization in mice overexpressing angiopoietin-1. Science. 1998;282:468 471. 7. Cho CH, Kammerer RA, Lee HJ, et al. COMP-Angl: A designed angiopoietin-1 variant with nonleaky angiogenic activity. Proc N~t! Ac~c! Sci U S A. 2004;101:5547~552. 8. Cho CH, Kammerer RA, Lee HJ, et al. Designed angiopoietin-1 variant, COMP-Angl, protects against radiation-induced endothelial cell apoptosis. Proc N~tl Acid Sci U S A. 2004;101:5553 5558. 9. Cho CH, Kim KE, Byun J, et al. Long-term and sustained COMP-Angl induces long-lasting vascular enlargement and enhanced blood flow [published correction appears in Circ Res. 2006;98:e69]. Circ Res. 2005;8:86 94. 10. Lee S, Kim W, Moon SO, et al. Renoprotective effect of COMP-angiopoietin-1 in db/db mice with type 2 diabetes. Nephrol Di~l Transplant. 2007;22:396 408. 11. Vinik AI, Park TS, Stansberry KB, Pittenger GL. Diabetic neuropathies. Di~betologi~. 2000;43:957 973. 12. Boulton AJ, Vinik AI, ArezzoJC, et al, for the American Diabetes Association. Diabetic neuropathies: A statement by the American Diabetes Association. Diabetes C~re. 2005;28:956 962. 13. Siemionow M, Demir Y. Diabetic neuropathy: Pathogenesis and treatment. J Reconstr Nicrosurg. 2004;20:241 252. 14. Dyck pJ, Davies JL, Wilson DM, et al. Risk fiactors for severity of diabetic polyneuropathy: Intensive longitudinal assessment of the Rochester Diabetic Neuropathy Study cohort. Diabetes C~re. 1999;22:1479 1486. 15. Dyck pJ, Zimmerman BR, Vilen TH, et al. Nerve glucose, fructose, sorbitol, myo-inositol, and fiber degeneration and regeneration in diabetic neuropathy. N EnglJ Ned. 1988;319:542~48. 16. Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. N~ture. 2000;404:787~90. 17. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulindependent diabetes mellitus. N EnglJ Ned. 1993;329:977 986. 18. Tomlinson DR, Willars GB, Carrington AL. Aldose reductase inhibitors and diabetic complications. Ph~rm~col Ther. 1992;54:151 194. 19. Yagihashi S, Kamijo M, Baba M, et al. Effect of aminoguanidine on functional and structural abnormalities in peripheral nerve of STZ-induced diabetic rats. Diabetes. 1992;41:47~2. 20. Nakamura J, Kato K, Hamada Y, et al. A protein kinase C-beta-selective inhibitor ameliorates neural dysfunction in streptozotocin-induced diabetic rats. Diabetes. 1999;48:2090 2095. 21. Vincent AM, Russell JW, Low P, Feldman EL. Oxidative stress in the pathogenesis of diabetic neuropathy. Endocr Rev. 2004;25:612 628.
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22. Cameron NE, Cotter MA. Neurovascular dysfunction in diabetic rats. Potential contribution of autoxidation and free radicals examined using transition metal chelating agents. J Clin Invest. 1995;96:1159 1163. 23. Suri C, Jones PF, Patan S, et al. Requisite role of angiopdetin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 1996;87:1171 1180. 24. Schmelzer JD, Zochodne DW, Low PA. Ischemic and reperfusion injury of rat peripheral nerve. Proc Nat! Acad Sci U S A. 1989;86:1639 1642. 25. Kurose I, Granger DN. Evidence implicating xanthine oxidase and neutrophils in reperfusioninduced microvascular dysfunction. Ann N Y Acad Sci. 1994;723:158 179. 26. Kim W, Moon SO, Lee SY, et al. COMP-angiopoietin-1 ameliorates renal fibrosis in a unilateral ureteral obstruction model. J Am Soc NephroL 2006;17:2474 2483. 27. Kim I, Moon SO, Park SK, et al. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression. Cin Res. 2001;89:477 479. 28. Nakae M, Kamiya H, Naruse K, et al. Effects of basic fibroblast growth i~actoron experimental diabetic neuropathy in rats. Diabetes. 2006;55:1470 1477. 29. Schratzberger P, Walter DH, Rittig K, et al. Reversal of experimental diabetic neuropathy by VEGF gene transfer.J Clin Invest. 2001;107:1083 1092. 30. Kato N, Nemoto K, Nakanishi K, et al. Nonviral gene transfer of human hepatocyte growth i~actor improves streptozotocin-induced diabetic neuropathy in rats. Diabetes. 2005;54:846 854.
ADDRESS CORRESPONDENCE T O : Tae Sun Park, MD, PhD, Endocrinology and Metabolism and D e p a r t m e n t of Internal Medicine, C h o n b u k National University Medical School, 634-18, K e u m - A m Dong, Jeonju, 561-712, South Korea. E-mail: pts@ chonbuk.ac.kr
3SS
Effect of Cartilage Oligomeric Matrix Protein Angiopoietin-1 on Peripheral Nerves in db/db Diabetic Mice Heung Yong Jin, MD1; Ming Han Piao, MD1; Ji Hyun Park, MD1; Hong Sun Baek, MD1; Sik Lee, MD2; Won Kim, MD2; Sung Kwang Park, MD2; Chong Hwa Kim, MD3; Gou Young Koh, MD4; and Tae Sun Park, MD, PhD 1
1Endocrinology and Metabolism and Department of Internal Medicine, Research Institute of Clinical Medicine, Chon&k National UniversiU Medical School,ffeonju, South Korea; eRenal Regeneration Laboratory and Department of Internal Medicine, Research Institute of Clinical Medicine, Chonbuk National University Medical School,ffeonju, South Korea; 3Bucheon Sejong Hospital, Bucheon, South Korea; and 4Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea
ABSTRACT B A C K G R O U N D : Vascular and inflammatory processes have been reported to be factors
in the pathogenesis of diabetic neuropathy. Angiopoietin-1 (Angl) plays essential roles in regulating vascular growth, development, maturation, permeability, and inflammation. O B J E C T I V E : The aim of this study was to investigate the effect of cartilage oligomeric matrix protein (COMP)-Angl, which is a soluble, stable, potent Angl variant, on peripheral nerves in db/db diabetic mice. M E T H O D S : The db/db diabetic mice were randomized into 2 groups based on their weight and glucose level and treated with recombinant adenovirus (Ade), expressing either COMP-Angl or the ~-galactosidase gene (LacZ) (control), for 8 weeks. Immunohistochemistry was performed using a polyclonal antibody of antiprotein gene product and a secondary antibody. Intraepidermal nerve fiber density (IENFD) was quantified as nerve fiber abundance per unit length of epidermis (IENF/mm). In addition, the total capillary length (TCD per unit length of epidermis was summed (mm/mm2). All slides were coded and the capillary length and the number of nerve fibers were calculated by a blinded observer. R E S U L T S : Ten diabetic db/db mice (mean [SD] weight, 38.7 [1.95] g) were randomized to receive Ade-COMP-Angl or Ade-LacZ. IENFD was significantly greater in the Ade-COMP-Angl group compared with the Ade-LacZ group (mean [SD] 8.95 [3.30] vs 3.57 [0.73]/mm; P < 0.05). TCL was also significantly greater in the Ade-COMP-Angl group (2.79 [0.99] vs 2.04 [0.58] mm/mm2; P < 0.05). Compared with baseline, fasting blood glucose concentration after 8 weeks of treatment decreased significantly more in the Ade-COMP-Angl group than in the Ade-LacZ group (489 [45] to 361 [81] vs 495 [48] to 521 [70] mg/dL; P < 0.05). Accepted~r pub/ication March 27, 2008. © 2 0 0 8 Excerpta Medica Inc. All rights reserved.
doi:10.1016/j.curtheres.2008.08.002 0011-393X/$32.00
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CONCLUSIONS: These results suggest that Ade-COMP-Angl might have had proliferative effects on peripheral nerve and cutaneous capillaries in this small animal study. Further investigation of the metabolic effect, target site, and related mediator of COMP-Angl is needed. (Curr Ther Res C/in Exp, 2008;69:343 355) © 2008 Excerpta Medica Inc. K E Y W O R D S : COMP-angiopoietin-1, db/db diabetic mice, peripheral neuropathy.
INTRODUCTION
Despite the fact that peripheral neuropathy is a serious microvascular complication and a leading cause of foot amputation, which increases the mortality rate of diabetic patients, many study outcomes attempting to clarify pathogenic mechanisms or to successfully treat diabetic neuropathy have been unsatisfactory) Because hyperglycemia is a major factor in the development of diabetic neuropathy, strict control of blood glucose concentration is an essential strategy in the prevention and treatment of this condition. 2,3 Inflammation is increased in diabetes, and enhanced inflammatory response can cause nerve injury in experimental neuropathy. 4 In addition, neural cell degeneration and decreased nerve blood flow have been reported to be pathophysiologic features of diabetic neuropathy. 5 Treatment with growth factors and anti-inflammatory agents that prevent neural cell degeneration and improve blood flow directly or indirectly might be considered an effective therapeutic approach. In addition to promoting angiogenesis, angiopoietin-1 (Angl) is known to stabilize endothelium and to have anti-inflammatory effects6; however, it is unclear whether Angl also has a beneficial effect on peripheral nerve growth related to cutaneous vasculature in the diabetic animal model. Cartilage oligomeric matrix protein (COMP)-Angl is an Angl variant that is reported to be a more effective therapeutic agent than native Angl in the way of efficiency, generation, and potency.7,8 For systemic treatment with COMP-Angl, 2 adenoviral vectors were developed that encode the COMP-Angl gene and the ~-galactosidase gene (LacZ). By delivering adenovirus (Ade)-COMP-Angl using a viral vector, long-term, sustained COMP-Angl circulation can be maintained. 9 This study investigated the neuroprotective effect of Ade-COMP-Angl, a soluble, stable, potent Angl variant, by estimating intraepidermal nerve fiber density (IENFD) and cutaneous vascularity in db/db diabetic mice. MATERIALS COMP-ANG1
AND AND
METHODS
ADE-COMP-ANG1
PREPARATION
Ade-COMP-Angl was made from recombinant Chinese hamster ovary ceils. A recombinant adenovirus that could express COMP-Angl or LacZ was constructed using the pAdEasy vector system (Qbiogene, Inc., Irvine, California), as was done in previous studies .7,8 ANIMALS
Male rib/rib mice were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed in our pathogen-free animal facility; 8- to 10-week-old mice were used for this
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study. The animal care protocol and the experimental procedures were approved by the Institutional Animal Care and Use Committee of the Chonbuk National University Medical School (Jeonju, South Korea). STUDY
DESIGN
For this study, rib/rib mice that were similar in weight and blood glucose concentration were randomized based on weight and glucose level into 2 groups, and 109 plaque-forming units of Ade-COMP-Angl or Ade-LacZ diluted in 50 laL of sterile 0.9% sodium chloride was injected intravenously through the tail vein for adenoviral treatment. Serum concentrations of Ade-COMP-Angl remained at elevated concentrations for 2 weeks, as previously described. 1° Consequently, Ade-COMP-Angl or AdeLacZ was injected 4 times at 2-week intervals. In a preliminary study, we found that LacZ had no effect on weight, blood glucose concentration, or other measured parameters in the same animal model. Therefore, LacZ was used as the control agent. Weight, blood glucose concentration, and food intake were measured at baseline and at 2-week intervals. Blood glucose concentration was measured in a sample drawn from a tail vein using Precision Xtra Plus ® (Abbott Laboratories, MediSense Products, Bedford, Massachusetts), and serum insulin concentration was analyzed using a radioimmunoassay (Neodine Laboratories, Seoul, South Korea). After 8 weeks of treatment, the mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and a punch biopsy of skin 3 m m in diameter was performed on the dorsum of both feet to quantify the degree of innervation and vascularity. IMMUNOHISTOCHEMISTRY
For immunohistochemistry on frozen microtome sections, skin tissue specimens were fixed with periodate-lysine-paraformaldehyde (2 % paraformaldehyde, 0.075 M lysine, 0.05 M phosphate buffer p H 7.4, 0.01 M sodium m-periodate) solution for 24 hours. After thorough rinsing in phosphate-buffered saline (PBS), which contained 20% glycerol and 0.1 M phosphate buffer, for 48 hours at 4°C, tissue specimens underwent cryoprotection with Tissue-Tec ® (OCT compound) (Miles, Elkhart, Indiana). Sections 40 lam were cut on a sliding microtome (Leica CM 1510 ®, Leica Microsystems AG, Wetzlar, Germany) perpendicular to the dermis and were immerged in PBS for 15 minutes at room temperature. Samples were then transferred into microtubules containing Dako Protein Block Serum-Free ® (Dako Corporation, Carpinteria, California) as a blocking buffer supplemented with 3% goat serum. After 30 minutes in the blocking buffer on a shaker table at room temperature, sectioned specimens were washed with PBS twice for 10 minutes and then incubated separately overnight at 4°C in 1 of 2 primary antibodies rabbit antiprotein-gene-product 9.5 (PGP 9.5) (Biogenesis, Poole, United Kingdom) for peripheral nerves at a dilution of 1:100 or hamster antimouse CD31 (1:100) (Chemicon International Inc., Temecula, California) for capillary endothelial ceils. The antibodies were diluted in antibody diluent (Dako Corporation) supplemented with 1% goat serum. After a sterile washing, the relevant secondary a n t i b o d i e s ~ o a t antirabbit immunoglobulin G (IgG)-FITC (1:200) or Cy3 Aff]niPure goat antiarmenian hamster IgG (heavy and light chain) (1:200) (Jackson
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ImmunoResearch Laboratories Inc., West Grove, Pennsylvania) were separately loaded (ie, immunostained) for 1 hour at room temperature in a dark room. After washing with PBS as above, sections were placed on slides and prepared with fluorescent mounting medium (Dako Corporation). QUANTIFICATION
OF INNERVATION
AND
CAPILLARY
VASCULARITY
PGP 9.5 immunoreactive nerve fibers in the epidermis of each section were counted at a magnification of 100x. Each individual nerve fiber with branching points inside the epidermis was counted as 1 fiber. For epidermal nerve fibers with branching points inside the dermis, each individual nerve fiber was counted separately. Conventional IENFD was derived and expressed as epidermal nerve fiber abundance per unit length of the epidermis (IENFD/mm). IENFD was calculated by a blinded observer, and all biopsy slides were coded and mixed with a set of normal slides to ensure examiner blinding. Photomicrographs of representative sections were captured using a digital camera (Axiocam HRC ®, Carl Zeiss, Goettingen, Germany). Capillary vascularity was also observed (by a blinded investigator) using the same method that was used to determine IENFD, including the photomicrographs. Images were collected and analyzed using a microscope (Axioskop2, Carl Zeiss Ltd., Welwyn City, United Kingdom) and the Axiovision 5.1 program (Carl Zeiss Ltd.). STATISTICAL
ANALYSIS
A power analysis (90%) was performed to determine sample size. Based on the power analysis, 5 mice per group were needed to detect significant between-group differences. Data were presented as mean (SD). The unpaired t test was used to compare the AdeLacZ and Ade-COMP-Angl groups. P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 12.0 software (SPSS Inc., Chicago, Illinois). RESULTS ANIMALS
Five mice (mean [SD] weight, 38.7 [1.95] g) were assigned to each of the 2 groups. EFFECTS
OF ADE-COMP-ANG1
ON CHARACTERISTICS
OF
dbldb M I C E
Characteristics of db/db mice after IV administration of Ade-LacZ or Ade-COMPAngl for 8 weeks (4 injections at 2-week intervals) are presented in Table I. Before treatment (baseline), the mice in the 2 groups did not differ significantly in age (data not shown), weight, food intake, or fasting blood glucose concentration. Although there was no significant difference in food intake between the groups during the experimental period, both body weight (51.8 [1.7] vs 54.1 [2.2] g; P < 0.05) and fasting blood glucose concentration (361 [81] vs 521 [70] mg/dL; P < 0.05) were significantly lower in the Ade-COMP-Angl group compared with the Ade-LacZ group. However, there was no significant between-group difference in serum insulin concentration after treatment (39.5 [5.1] vs 39.7 [3.9] laU/mL) (Table I).
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H.Y. JIN ET AL.
Table I. Characteristics of the diabetic db/db mice at baseline and after 8 w e e k s of treatment (N = 1 0 ) . Data are mean (SD).
Ade-COM P-Angl (n = 5) Characteristic Weight, g Food intake, g / 2 4 h
Ade-LacZ (n = 5)
Baseline
Week 8
Baseline
38.6 (2.1)
51.8 (1.7)*
38.8 (1.9)
54.1 (2.2)
7.8 (0.8)
8.2 (0.7)
7.8 (1.1)
Serum insulin, pU/mL
8.3 (0.5) 39.5 (5.1)
Fasting blood glucose, mg/dL
4 8 9 (45)
Week 8
39.7 (3.9)
361 (81)*
495 (48)
521 (70)
IENFD, no./mm
8.95 (3.30)*
3.57 (0.73)
Total capillary length per unit length of epidermis, mm/mm 2
2.79 (0.99)*
2.04 (0.58)
Ade = adenovirus; COMP = cartilage oligomeric matrix protein; Angl = angiopoietin-1; LacZ : ~-galactosidase gene; IENFD= intraepidermal nerve fiber density. *P < 0.05 versus Ade-LacZ-treated group. E F F E C T S OF A D E - C O M P - A N G 1
ON I N T R A E P I D E R M A L
NERVE FIBERS
Mean (SD) IENFD was significantly greater (>2-fold) in the Ade-COMP-Angl group compared with the Ade-LacZ group (8.95 [3.30] vs 3.57 [0.73]/mm; P < 0.05). IENFD of each mouse is shown in Table II. The morphologic findings o f l E N F D in each group appeared to increase (Figure 1). E F F E C T S OF A D E - C O M P - A N G 1
ON T O T A L C A P I L L A R Y
LENGTH
Mean (SD) total capillary length (TCL) was also significantly greater in the AdeCOMP-Angl group than in the Ade-LacZ group (2.79 [0.99] vs 2.04 [058] ram/ram2; P < 0.05). TCL for each mouse is shown in Table II; the increased pattern ofvascularity Table II. Intraepidermal nerve fiber density (IENFD) and total capillary length (TCL) of the individual diabetic db/db mice after 8 w e e k s of t r e a t m e n t with adenovirus (Ade)-cartilage oligomeric matrix protein-angiopoietin-1 ( C O M P - A n g l ) or A d e - ~ - g a l a c t o s i d a s e gene (LacZ). Data are mean (SD).
Ade-COM P-Angl-Treated IENFD Mouse Mouse Mouse Mouse Mouse
1 2 3 4 5
Mean (SD)
6.18 12.52 8.98 7.32 9.76
(1.23) (6.37) (3.71) (2.36) (4.94)
8.95 (3.30)
TCL 3.33 2.72 2.88 2.18 2.85
(0.72) (0.57) (0.71) (0.38) (2.61)
2.79 (0.99)
Ade-LacZ-Treated IENFD 3.22 4.88 3.32 3.17 3.29
(0.74) (1.74) (2.17) (0.63) (1.22)
3.57 (0.73)*
TCL 2.36 1.83 2.07 1.95 2.01
(0.59) (0.62) (0.54) (0.56) (0.61)
2.04 (0.58)
*P < 0.05 versus Ade-COMP-Angl-treated group.
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CURRENT THERAPEUTIC RESEARCH
Figure 1. Intraepidermal nerve fiber density (IENFD) in db/db diabetic mice after 8 weeks of treatment with (A) adenovirus (Ade)-carUlage oligomeric matrix protein-angiopoietin-1 (COMP-Angl) or (B) Ade-~-galactosidase gene (LacZ). Arrows indicate the peripheral nerves maintained in the epidermis, which was branched in the dermis (200X); capillary endothelial immunostaining with hamster antimouse CD31. (continued)
348
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in the Ade-COMP-Angl group is presented in schematic fashion in Figure 2 (including photomicrographs). DISCUSSION
Diabetic neuropathy is one of the most important factors affecting the prognosis of diabetic patients. Approximately half of diabetic patients are reported to have neuropathic symptoms. 11 In particular, significant morbidity among diabetic patients is associated with sensory deficit of the lower extremities, potentially resulting in foot ulceration or amputation. Prevention and treatment of diabetic neuropathy have often been unsatisfactory. The effects of various agents on a hypothesized pathogenic cause of
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Figure 2. Cutaneous capillary vascularity shown in schematic representations of skeletonized microvessels of db/db diabetic mice after 8 weeks of treatment with (A) adenovrius (Ade)-cartilage oligomeric matrix protein-angiopoieUn-1 (COMP-Angl) and (B) Ade-13-galactosidase gene (LacZ) (200X; capillary endothelial immunostaining with hamster antimouse CD31). (continued)
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diabetic neuropathy have been investigated in both experimental and clinical trials. Most of these agents have been found to have promising results in experimental studies, but the findings in clinical trials have been less convincing. 12,13 Several factors, including hyperglycemia, the polyol pathway, protein kinase C (PKC), and nonenzymatic glycation have been postulated to be involved in the pathogenesis of diabetic neuropathy. 14 16 Most therapeutic agents for the treatment of diabetic neuropathy have been focused on these putative mechanisms. 17 21 Reports suggest that vascular factors, including microangiopathy or endoneural ischemia, and neuropathic processes may be associated with diabetic neuropathy. 2°,22 In addition, studies have found that increased inflammation in diabetes can cause peripheral nerve injury.4 New anti-inflammatory strategies targeting both the neurotrophic and angiogenic aspects of diabetic neuropathy may be possible therapies. Angl is a ligand of the Tie2 receptor, which is a member of the tyrosine kinase family that is expressed on vascular endothelial ceils. Angl, which is mediated by phosphorylating the Tie2 receptor and signaling via the Akt pathway, has therapeutic potential in angiogenesis and vascular endothelial dysfunction; however, Angl has aggregation and insolubility problems due to the superclustering domain and multimerization of Angl. 23 For these reasons, a chimeric form of Angl with COMP (COMP-Angl) was developed to avoid these limitations. COMP-Angl is a soluble, stable, potent Angl variant that has been found to be effective in preventing endothelial dysfunction and inflammation.7,8 However, the effect of COMP-Angl on peripheral neuropathy in diabetes has not yet been studied in experimental models. Therefore, we investigated the effects of COMP-Angl on peripheral nerves of,rib/rib diabetic mice. Inflammatory mediators have been reported to be pathogenic mechanisms of diabetic neuropathy. 24,25 We reported that the anti-inflammatory effect of COMPAngl involves inhibiting the nuclear factor (NF)-KB pathway, which subsequently decreases activation of transforming growth factor (TGF)-~. 1° We also reported that COMP-Angl could increase blood flow by improving endothelial survival and vascular size. 26'27 Vascular factors related to tissue blood flow that can be targeted for treatment in diabetic peripheral neuropathy include increasing endothelium-derived relaxing factor, inhibition of PKC, suppression of NF-KB, and modulation of vascular endothelial growth factor (VEGF) subtype. Several factors that have an angiogenic effect (eg, VEGF, hepatocyte growth factor, fibroblast growth factor) have been reported to have therapeutic potential in diabetic neuropathy. 28 30 Neurotrophic factors, such as nerve growth factor, have been studied in animal models of diabetic neuropathyS; however, the clinical usefulness of this therapy was limited by the absence of an established therapeutic dose and reliable data. In this study, the effect of COMP-Angl might not have been mediated by direct action on peripheral nerves; rather, COMP-Angl may have triggered the activation of a cytokine cascade involved in nerve regeneration or inhibition of nerve degeneration in association with angiogenesis. Although previous studies have identified the effect of COMP-Angl on cytokines, a variety of cytokines will need to be investigated (including histologic analysis) to clarify the mechanism of action of COMP-Angl in angiogenesis and nerve regeneration in diabetic neuropathy. 8 10
352
H.Y. JIN ET AL.
In this study, fasting blood glucose concentration was significantly lower in db/db mice treated with Ade-COMP-Angl compared with those treated with Ade-LacZ. However, the serum insulin concentration was not significantly difterent between the 2 groups. These results suggest that the insulin-sensitizing eftect and resulting metabolic eftects of COMP-Angl might also contribute to neuroprotection in diabetic mice. Therefore, the metabolic eftect of COMP-Angl in diabetic mice, including its exact mechanism of action, and another group showing similar glucose control with COMPAngl warrant further investigation. In our study, IENFD and TCL were greater in the group treated with Ade-COMPAngl compared with the Ade-LacZ group. An increased number of nerve fibers and enriched vascularity were observed in the series of photomicrographs in the AdeCOMP-Angl group. LIMITATIONS
Our study had several limitations. The effects of COMP-Angl on peripheral nerves and capillaries might be related to its glucose-lowering effects. Next, if IENFD and TCL in both groups were measured prior to treatment and compared with our findings after treatment, it would have been more evident whether COMP-Angl indeed had a proliferative effect on diabetic peripheral neuropathy. Therefore, we cannot definitively conclude that COMP-Angl protected the mice from any subtle loss of nerve fibers or vascularity or that it was associated with improvement in these factors when compared with LacZ. Finally, the presence of COMP-Angl receptors in the peripheral nerves and the related mediators such as TGF-~ or NF-KB, were not investigated in this study (except to observe the morphologic changes), which would reveal more precise evidence on COMP-Angl effects. CONCLUSIONS
These db/db diabetic mice treated with Ade-COMP-Angl had greater IENFD and cutaneous capillary length compared with the Ade-LacZ group in this small animal study. We suggest that Ade-COMP-Angl might have therapeutic potential, with a dual impact on peripheral nerves and cutaneous capillaries in this diabetic animal model. Ade-COMP-Angl treatment might also improve blood glucose concentration. Further studies targeting molecular markers and COMP-Angl receptors in peripheral nerves are warranted to determine the direct eftect of COMP-Angl on neural ceils. ACKNOWLEDGMENTS
This work was supported in part by grants from the National Research Laboratory Program of Korea Science and Engineering Foundation (Jeonju, South Korea) and the Medical Science Education Program for the 21st Century of Chonbuk National University Medical School. REFERENCES
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ADDRESS CORRESPONDENCE T O : Tae Sun Park, MD, PhD, Endocrinology and Metabolism and D e p a r t m e n t of Internal Medicine, C h o n b u k National University Medical School, 634-18, K e u m - A m Dong, Jeonju, 561-712, South Korea. E-mail: pts@ chonbuk.ac.kr
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