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Anesth Analg 2008; 107:686-692
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e31817e6719
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ANALGESIA

Prolonged Use of High-Dose Morphine Impairs Angiogenesis and Mobilization of Endothelial Progenitor Cells in Mice

Chen-Fuh Lam, MD, PhD*, Pei-Jung Chang, MD*, Yu-Sheng Huang, MD*, Yen-Hui Sung, MD*, Chien-Chi Huang, BS*, Ming-Wei Lin, MS*{dagger}, Yen-Chin Liu, MD, MS*, and Yu-Chuan Tsai, MD*

From the *Department of Anesthesiology; and {dagger}Institute of Basic Medical Sciences, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan.

Address correspondence and reprint requests to Yu-Chuan Tsai, MD, Department of Anesthesiology, National Cheng Kung University, Medical College and Hospital, Tainan City, Taiwan. Address e-mail to yctsai{at}mail.ncku.edu.tw.

Abstract

BACKGROUND: Morphine is one of the most commonly prescribed analgesics for treating wound pain. Using a mouse model of excisional wound injury, we determined the effects of high-dose morphine on angiogenesis and mobilization of endothelial progenitor cells.

METHODS: An excisional wound was created on mice treated with placebo or morphine (20 mg/kg, i.p. injection for 14 days). Wound healing was compared by measuring the final-to-initial wound area ratio. Generation of superoxide anions in the wound was determined by luminol-enhanced chemiluminescence. Circulating mononuclear cells were isolated and measured for endothelial progenitor cell (defined as CD34+/CD133+ cell) counts. In vivo and in vitro measurements of angiogenesis after morphine treatment were performed using the Matrigel assay.

RESULTS: Mice treated with morphine had reduced wound closure and higher wound superoxide ions concentrations than control mice. Morphine reduced the number of postwound circulating endothelial progenitor cells. Matrigel assay showed impaired angiogenesis in animals and reduced capillary tube formation in cultured endothelial cells treated with morphine.

CONCLUSION: High-dose morphine impaired angiogenesis, increased systemic oxidative stress, and impaired mobilization of endothelial progenitor cells. This study emphasizes the potential detrimental effect of high-dose morphine on angiogenesis after systemic administration.

High-dose morphine is commonly used for the control of severe wound pain. High-dose morphine has cytotoxic and pro-oxidant effects in hepatocytes,1 macrophages,2 glomerular mesangial and epithelial cells,3,4 and vascular endothelial cells.5 We have recently shown that high-dose morphine impairs endothelial function by activation of vascular NADPH oxidase and increased production of superoxide anions.6 Since vascular endothelial function is well correlated with angiogenesis and mobilization of endothelial progenitor cells, the pro-oxidant effects of morphine after systemic administration might affect angiogenesis.

Angiogenesis is the process of expansion of the capillary network or formation of new vasculature from preexisting vessels, which is essential in tissue repair and regeneration. In mice with endothelial nitric oxide synthase deficiency, impaired angiogenesis was shown by reduced capillary ingrowth into subcutaneously implanted Matrigel plugs. The reduced angiogenesis was associated with delayed wound healing.7 However, very few studies are available investigating the effect of morphine on angiogenesis, and results are controversial.8,9

The discovery of the circulating or bone marrow- derived endothelial progenitor cells during the past decade has altered our understanding of postnatal vasculogenesis and angiogenesis.10 Mobilization of endothelial progenitor cells into systemic circulation contributes significantly to angiogenesis during tissue regeneration.11 Transplantation of day-7 endothelial progenitor cells significantly accelerates the wound repair process by facilitating neovascularization in a murine dermal excisional wound model.12 Using a mouse model of excisional wound injury and Matrigel assays, we tested the hypothesis that high-dose morphine impairs angiogenesis. We also investigated the effect of high-dose morphine on the mobilization of endothelial progenitor cells.

METHODS

Mouse Model of Wound Healing
After approval by the Institutional Animal Care and Use Committee (The National Cheng Kung University, Tainan, Taiwan), mice (C57BL/6J, 8–10 wk old) were obtained from the Animal Center of the National Cheng Kung University. The animals were housed at a controlled temperature of 21 ± 0.5°C in wire-mesh cages, with free access to food and water. After being anesthetized by IM injection of ketamine (30 mg/kg), a full-thickness excisional wound (approximately 2 cm in diameter) was created on the dorsum of mice along the midline by a surgical scissors. The wound was covered by a transparent dressing (3M Health Care, St Paul, MN).7

After the surgical procedures, animals were randomly assigned to control or morphine-treated group and received intraperitoneal injection of normal saline or morphine (20 mg · kg–1 · d–1, the National Bureau of Controlled Drugs, Department of Health, Taipei, Taiwan) for 14 days, as described in our previous study.6,13 The dose of morphine (20 mg/kg) has been shown to produce a significant analgesic effect with clinically relevant serum concentrations of morphine and its metabolites (morphine-3-gluronide) in mice.14–16 This dose of morphine has also been used in mouse and rat models of morphine dependence.1,17–19

At the end of the treatment, mice were killed by injection of pentobarbital (250 mg/kg, i.p.). Excisional wounds were photographed and areas of the wound were measured using TwinCAD software (version 3.2, TCAM Development Inc., Bellevue, WA). Wound healing was measured as the ratio of final-to-initial wound areas. Wound tissues were excised for assay.

Physiological Responses and Blood Analysis
Physiological variables, including body weight, water consumption, and chow consumption were recorded. Arterial blood samples were collected by direct cardiac puncture. Hemoglobin and blood glucose levels were analyzed with a blood gas analyzer (GEM Premier 3000, Instrumentation Lab, Lexington, MA).

Measurement of Superoxide Anions
The release of superoxide anions in the wound was measured using chemiluminescence assay as previously described.20,21 Excised wound tissues were immersed in Krebs-HEPES buffer solution (in mmol/L: NaCl 135.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, MgSO4 1.2, EDTA 0.026. glucose 10.1, and HEPES 20; pH 7.4) for 30 min at 37°C. The tissues were then transferred into the luminol-enhanced chemiluminescence chamber. The release of superoxide was assessed by a chemiluminescence analyzer (Tohoku Electronic CLA 2100, Sendai, Japan). The relative chemiluminescence densities were normalized to the dry weight of the tissue. The final concentration of luminol (Sigma-Aldrich, St. Louis, MO) was 2 x 10–5 M.

To access the production of superoxide anions in the systemic circulation, peripheral blood mononuclear cells were isolated by density gradient centrifugation with Ficoll-Plaque Plus (Amersham Biosciences, Buckinghamshire, UK). Isolated mononuclear cells were suspended in phosphate buffered saline and incubated with dihydroethidium (5 µmol/L, BD Transduction Labs, San Jose, CA) for 15 min at 37°C. The mononuclear cell suspension was washed with phosphate buffered saline and fixed in 1% paraformaldehyde. Dye oxidation was measured using a FACScan flow cytometer (BD Biosciences, San Jose, CA).20,22

Measurement of Circulating Endothelial Progenitor Cells
Isolated mononuclear cells were resuspended in 100 µL of a fluorescence-activated cell sorting buffer containing phosphate buffered saline, 0.1% bovine albumin, and aprotinin. Cells were stained with fluorescent conjugated antibodies for CD34 (BD Biosciences, San Jose, CA) and CD133 (Miltenyi Biotec GmbH, Gladbach, Germany). CD34 and CD133 have been well recognized as the specific markers of circulating endothelial progenitor cells.23 Mouse IgG1, {kappa}-isotype antibody (BD PharmingenTM) was used as a control antibody. Cell fluorescence was measured immediately under a flow cytometry and expressed as relative mean fluorescence intensity. In this particular experiment, a blood sample was also drawn from mice without an excisional wound in order to identify the direct effect of high-dose morphine (20 mg · kg–1 · d–1 for 14 days) on the mobilization of endothelial progenitor cells.

Murine Angiogenesis Assay
To assess angiogenesis in vivo, 500 µL of Matrigel (BD Bioscience, San Jose, CA) containing fibroblast growth factor (10 ng/mL, R&D System, Minneapolis, MN) and heparin (60 U/mL, B.Braun Melsungen AG, Melsungen, Germany) was injected subcutaneously into the abdominal wall of mice 11 days after the commencement of morphine or saline administration (as described above).24 Three days after Matrigel injection, the mice were euthanized and the Matrigel plug was excised. Angiogenesis was assessed by the expression of a specific endothelial cell marker CD31 (monoclonal anti-CD31 antibody; BD Pharmigen, San Diego, CA) on the Matrigel plug.24 Briefly, the unfixed frozen Matrigel plugs were sectioned (10–15 µm in thickness) with a cryostat and incubated with the anti-CD31 antibody for 2 h at room temperature. After washing with phosphate buffered saline, the sectioned tissues were then incubated with FITC-conjugated secondary antibody for 30 min. The expression of green fluorescence on the Matrigel sections was detected under a laser scanning confocal imaging system (10x). The levels of fluorescent density on each section were quantified using the AlphaEase FCTM software (Alpha Innotech Corporation, San Leandro, CA).

In vitro Capillary Tube Formation Assay
Growth factor-free Matrigel was added onto the 24-well plates (400 µL/well). Human umbilical vein endothelial cells were cultured in the Matrigel-coated wells at a density of 4.8 x 104 cells/well with the supplement of endothelial basal medium-2 (EBM-2, Cambrex, Baltimore, MD) in a 95% air-5% CO2 environment at 37°C for 24 h. Capillary tube formation was quantified by counting the pixels of tubules from three randomly selected fields in each cultured well under microscopy (10x).25 Cells between passage five and eight were used in this study.

Statistical Analysis
Unless otherwise specified, the results are presented as mean ± sem. Data were compared by an unpaired t-test or analysis of variance, as appropriate. Statistical significance was accepted at a level of P < 0.05.

RESULTS

Effects of Morphine on Physiological Responses and Blood Analysis
Physiological responses and blood gas analysis of mice receiving placebo or prolonged treatment with high-dose morphine are shown as Table 1. There were no statistically significant differences in body weight gain or daily consumption of chow and water between the control and morphine-treated animals. Hemoglobin and blood glucose were also similar between the two groups.


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  		Table 1. Physiological Variables and Blood Analysis

 

Effects of Morphine on Healing of Excisional Wound
Compared with controls, the final-to-initial wound area ratio was significantly increased in mice treated with morphine (8.3% ± 2.2% vs 26.2% ± 6.9%, respectively; P = 0.026, n = 6 in each group), indicating that high-dose morphine delayed wound healing.

Effects of Morphine on Superoxide Production in Wound and Circulating Mononuclear Cells
Levels of superoxide anions were significantly elevated in the excised wound tissue (Fig. 1A), and in the isolated circulating mononuclear cells (Fig. 1B) in animals treated with morphine.


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  		Figure 1. Formation of superoxide anions in the excisional wound tissue (A) and circulating mononuclear cells (mononuclear cells) (B) after intraperitoneal administration of placebo or high-dose morphine. Levels of superoxide anions were quantified by luminol-enhanced chemiluminescence assay in the wound tissue (A), and intracellular levels of superoxide anions in the isolated mononuclear cells were measured by dihydroethidium assay under a flow cytometry (B). Both measurements indicate that high-dose morphine increases systemic and regional production of superoxide anions. *P < 0.05 in comparison to controls using unpaired t-test, and are shown as mean ± sem n = 6–8 different animals in each experimental group.

 

Effects of Morphine on Number of Circulating Endothelial Progenitor Cells
In the absence of an excisional wound, the administration of high-dose morphine did not affect the number of circulating endothelial progenitor cells (defined as CD34+/CD133+ mononuclear cells, Fig. 2). Creation of an excisional wound significantly enhanced the number of circulating CD34+/CD133+ mononuclear cells in the control group, and CD34+ mononuclear cells in the morphine-treated animals (Fig. 2), suggesting that wound healing was associated with mobilization of endothelial progenitor cells into the systemic circulation.11 However, in comparison to saline-treated controls, morphine treatment significantly suppressed the number of circulating epithelial progenitor cells (CD34+/CD133+ cells) after creation of an excisional wound (Fig. 2).


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  		Figure 2. Mobilization of bone marrow-derived CD 34+/ CD133+ endothelial progenitor cells after creation of an excisional wound and administration of morphine. Isolated mononuclear cells were suspended in fluorescence-activated cell sorting buffer. Cells were stained with fluorescent conjugated antibodies for CD34 and CD133, and fluorescence density was measured by a flow cytometry. Compared with controls, morphine significantly suppressed the number of circulating endothelial progenitor cells after creating an excisional wound. Data were analyzed by analysis of variance. #P < 0.05 compared with absence of wound within each group; *P < 0.05 compared with morphine group. n = 7–8 different animals in each experimental group.

 

Effects of Morphine on Angiogenesis
High-dose morphine significantly reduced the expression of CD31 on the Matrigel plugs (Fig. 3). Morphine decreased tube formation of cultured human umbilical vein endothelial cells on Matrigel in a concentration-dependent manner (Figs. 4A–D).


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  		Figure 3. Angiogenesis assay by examining the expression of FITC-conjugated CD31 in Matrigel plugs implanted subcutaneously into the mice receiving placebo (A) or morphine (B) injection for 14 days. The green fluorescence secondary antibody was detected by a laser scanning confocal imaging system (10x). Expression of CD31 (green fluorescence, arrows) was significantly reduced in the Matrigel excised from mice treated with high-dose morphine. *P = 0.006 compared with controls. Data were analyzed by unpaired t-test. n = 6 different animals in each experimental group.

 

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  		Figure 4. In vitro angiogenesis assay. Growth factor-free Matrigel was added onto the 24-well plates. Human umbilical vein endothelial cells were cultured with the supplement of endothelial basal medium-2 without and with morphine (10–10 to 10–4 M) at 37°C for 24 h. Capillary tube formation was quantified by counting the number of pixels from three randomly selected fields in each well under microscopy (10x). Morphine decreased tube formation in a concentration-dependent manner. Data were analyzed by analysis of variance and are presented as mean ± sem *P < 0.05 compared with control; n = 5 in triplicate. A: control, B to D: morphine 10–10 to 10–4 M, respectively.

 

DISCUSSION

This study provides in vivo and in vitro evidence that, in mice, systemic administration of high-dose morphine is associated with impaired mobilization of endothelial progenitor cells and angiogenesis, leading to impaired healing of excisional wounds. Systemic administration of high-dose morphine also increased local and systemic oxidative stress. These results underscore the potential anti-angionenic and pro-oxidant effects of high-dose morphine, which may affect tissue regeneration, wound healing, and the progression of cardiovascular disease.

There are very limited data demonstrating the effects of morphine on wound healing after parenteral administration. Using an open ischemic wound model in rats, Poonawala et al. group reported that topical application of morphine accelerated wound closure with enhanced expression of endothelial and inducible nitric oxide synthase.26 In contrast, Rook and McCarson recently demonstrated that topical use of morphine caused a significant, concentration-dependent delay in wound closure in a rat model of cutaneous excisional wound injury.27 The authors concluded that topical morphine affects wound healing by activating opioid receptors on the cutaneous afferent neurons, thereby inhibiting the release of neuropeptides, such as substance P and neurokinin A.

Wound healing is divided into four overlapping biomolecular phases: coagulation, inflammation, migration-proliferation, and remodeling.28 During the inflammatory phase, wounding initially results in a 60%–70% reduction in endogenous antioxidant activity, followed by a balanced oxidative homeostasis after 14 days.29 The major two cell types that contribute to the wound healing phase are the neutrophils and monocytes responsible for wound debridement and cell chemotaxis.30 Poor wound healing has been shown to be associated with prolonged status of excessive oxidative stress, leading to tissue damage.29 The damage is mediated by radical and nonradical reactive oxygen species, such as superoxide anions, hydroxyl radicals, and hydrogen peroxide. Therefore, levels of reactive oxygen species in the healing wound or circulating neutrophils are useful prognostic biomarkers in wound healing.31,32

Based on these previous studies, we examined the effects of morphine on wound healing 14 days after administration, when a balanced oxidative homeostasis is normally re-established. By measuring body weight gain, daily food and water intake, blood glucose, and hemoglobin, we assessed whether chronic administration of high-dose morphine would result in physiological stress, which in turn affects wound healing. Our results showed that prolonged administration of morphine up to 14 days did not produce detectable physiological changes in mice, indicating that the effect of morphine on wound healing is less likely to be derived from physiological stress, as measured in Table 1. After treatment with high-dose morphine, closure of excisional wounds in mice was significantly impaired. High-dose morphine treatment was also associated with increased levels of superoxide anions in the circulating mononuclear cells and wound tissues. In previous reports, increased systemic oxidative stress has been reported after large doses of parenteral administration of morphine.1,3–5 Since excessive oxidative stress is detrimental to the normal wound healing process,33,34 increased systemic and regional production of superoxide anions may thus serve as an important major molecular mechanism responsible for delayed wound healing in animals treated with high-dose morphine.

Previous studies have demonstrated that impaired angiogenesis is associated with delayed wound healing,7 whereas promotion of angiogenesis accelerates the healing process of wounds.35 Since their discovery by Asahara et al. in 1997,10 evidence continues to accumulate on the role of circulating endothelial progenitor cells in neovascularization and vasculogenesis. These cells are mobilized from bone marrow to the peripheral circulation by cytokines, growth factors, tissue injury, and ischemia.23 The endothelial regenerative potential of endothelial progenitor cells has been investigated in a variety of animal models, including hindlimb ischemia,36,37 myocardial ischemia,38,39 carotid artery injury,40 and vascular graft survival.41 Several clinical studies have reported encouraging outcomes in patients with critical limb ischemia or myocardial infarction after administration of endothelial progenitor cells.42–44 The animal and human studies suggest that circulating endothelial progenitor cells may function as an endogenous repair mechanism in forming new vessels by direct incorporation and paracrine-mediated effects.

In this study we found that creating an excisional wound in mice significantly enhanced the number of circulating CD34+/CD133+ mononuclear cells, reinforcing the notion that tissue regeneration during wound healing mobilizes endothelial progenitor cells. High-dose morphine attenuated the number of circulating endothelial progenitor cells. We tested the effect of morphine on circulating endothelial progenitor cells at baseline conditions. In the absence of an excisional wound, high-dose morphine did not affect the number of circulating endothelial progenitor cells, suggesting a lack of significant stimulating or inhibitory effects on the bone marrow. Alexander et al. reported a similar finding that morphine treatment alone did not alter splenic T-cell proliferation, whereas administration of morphine significantly suppressed T-cell proliferation in mice treated with burn injury.45

We tested the effects of morphine on angiogenesis using the in vivo and in vitro Matrigel assays. In cultured endothelial cells, morphine reduced capillary tube formation in a concentration-dependent manner. Levels of angiogenesis were compared by quantifying the expression of CD31, a biomarker of vascular endothelial cells, on the Matrigel plugs implanted subcutaneously in mice treated with or without morphine. Consistent with the in vitro experiment, fluorescent densities of CD31 in the isolated Matrigel plugs were also significantly reduced in mice treated with high-dose morphine. Our in vitro and in vivo experiments suggest that high-dose morphine impairs angiogenesis.

The literature is mixed concerning morphine and angiogenesis. Gupta et al. reported that morphine stimulates angiogenesis, especially in tumor angiogenesis, and enhances tumor growth.9 Other researchers have shown that high-dose morphine reduced blood vessel proliferation,46 or reduced variability and increased apoptosis of cultured endothelial cells.5 Furthermore, Roy et al. demonstrated that morphine (1–100 ng/mL) inhibited expression of vascular endothelial growth factor in endothelial cells and cardiac myocytes during hypoxic conditions in a concentration- dependent fashion.8,47 The authors concluded that the inhibitory effects of morphine on endothelial cells may suppress neovascularization of ischemic myocardium. Although in vitro studies on cultured endothelial cells provide important insights regarding the behavior of vascular endothelium in response to different concentrations of morphine,9,48 the extent to which these findings accurately reflect in vivo results is problematic, as the status of oxidation was ignored in the in vitro conditions. Nevertheless, consistent with the inhibitory effect of morphine on endothelial cells during hypoxia,8,47 our results indicate that, in the presence of increased oxidative stress and wounding, high-dose morphine was associated with impaired angiogenesis, and hence delayed healing of excisional wounds. Although we were not able to demonstrate direct relationships between impaired angiogenesis, oxidative stress, and mobilization of endothelial progenitor cells in our current experimental design, our data support previously published findings that these factors are highly inter-related.49,50

In conclusion, our current study demonstrated that the anti-angiogenic effects of systemic high-dose morphine associated with delayed would healing increased local superoxide anions production and impaired mobilization of endothelial progenitor cells into the systemic circulation.

Footnotes

C.-F. Lam and P.-J. Chang contributed equally to the study.

Accepted for publication April 15, 2008.

Supported by National Science Council, Taiwan grant NSC-94-2314-B-006-046 to YCT.

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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2008 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press