高劑量嗎啡通常一般是使用在臨床上癌症病人身上的止痛及手術後傷口疼痛的一個止痛劑。病人長期使用高劑量的嗎啡已經被知道與幾個複雜的心血管及組織的修復缺陷有關。因此這個研究主題主要探討高劑量嗎啡在血管內皮細胞功能、血管新生及傷口癒合的生物及分子機制的影響，使用容易對嗎啡上癮的小鼠及培養內皮細胞來做分析。
小鼠利用腹腔注射嗎啡每天每隻打20mg/kg連續打14天，控制組打生理食鹽水。之後，將主動脈分離出來測血管收縮的功能，利用西方墨點法測p47蛋白質及螢光顯微鏡偵測超氧化陰離子的表現。利用acetylcholine促使內皮細胞舒張在高劑量嗎啡治療的小鼠是顯著的被減少，另外，高劑量嗎啡治療的小鼠在螢光染色染dihydroethidium (DHE)也是增加的及p47的蛋白質表現也是較高的。
論文的第二部份，利用小鼠模式切除傷口探討在高劑量的嗎啡以及endothelial progenitor cells (EPCs)的動員，切除傷口的老鼠利用腹腔注射給予嗎啡(20mg/kg)連續給予14天，最後測量傷口面積大小復原程度，利用luminol冷光激發來偵測傷口超氧化陰離子的產生，測量被分離出來的單核球細胞的EPC (CD34+/CD133+ cell)的表現量。利用Matrigel分析在小鼠體內及體外嗎啡組在血管新生的結果，結果顯示傷口癒合的程度嗎啡組比控制組還要差，超氧化陰離子也產生的比較多，而且高劑量嗎啡也減少了切除傷口的EPC循環，Matrigel分析顯示，高濃度嗎啡治療組的動物血管新生能力降低，在內皮細胞的培養微血管組成減少。
全部研究證明一些新的發現。首先，高劑量的嗎啡減少血管內皮細胞的功能主要是經由血管內超氧化陰離子的增加產生，而NADPH oxidase的活化可能是經由內皮細胞NO的產生，以致於生物功能被減少所造成的分子機制。第二部份，高劑量嗎啡延緩切除傷口的癒合與血管新生減少的證明。這個抗血管新生的影響主要是超氧化陰離子的產生增加與EPC的循環被減少有關，從高劑量嗎啡治療的結果發現內皮細胞功能減少與血管新生及EPC的動員被減少而人類在嗎啡的治療下也可能導致增加心血管疾病。

High-dose morphine has been extensively used in the control of postoperative and cancer pain. Patients receiving prolonged administration of high-dose morphine are known to be associated with certain cardiovascular complications and tissue regeneration defects. This research thesis aims to investigate the biological effects and molecular mechanisms of high-dose morphine on the vascular endothelial function, angiogenesis and wound regeneration using murine models of morphine-dependence and cultured endothelial cell assays.
Mice were subjected to placebo or morphine (20 mg/kg, i.p.) injection for consecutive 14 days. Aortas were harvested for assessment of vasomotor function by isometric force recordings. Protein expression p47phox (a major subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase) was determined by Western blotting. Generation of superoxide anions was detected under confocal microscope. Endothelium-dependent relaxations to acetylcholine were significantly reduced in morphine-treated animals, but were normalized by superoxide scavenging. Fluorescent densities of dihydroethidium and expression of p47phox were increased in the aorta of morphine-treated mice.
In the second part of this thesis, the candidate determined the effects of high-dose morphine on angiogenesis and mobilization of endothelial progenitor cells (EPCs) in a mouse model of excisional wound injury. Excisional wound was created on control and morphine-dependent mice. 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 EPC (defined as CD34+/CD133+ cell) counts. In vivo and in vitro measurements of angiogenesis following morphine treatment were performed using the Matrigel assay. The results showed that wound closure was significantly reduced in mice treated with morphine when compared with controls, and higher levels of superoxide anions were generated in these wounds. High-dose morphine reduced numbers of circulating EPCs following creation of excisional wound. Matrigel assay showed impaired angiogenesis in animals and reduced capillary tube formation in cultured endothelial cells treated with high-concentration of morphine.
Collectively, this research thesis demonstrated a number of novel findings. First, high-dose of morphine impairs vascular endothelial function by increased production of vascular superoxide anions. Activation of NADPH oxidase may be the molecular mechanisms responsible for reduced bioavailability of endothelium-derived NO. Second, systemic administration of high-dose morphine delays healing of excisional wounds and impairs angiogenesis. This antiangiogenic effect is associated with increased superoxide anions production and impaired mobilization of EPCs. In line with direct endothelial dysfunction, impaired angiogenesis and EPC mobilization resulted from high-dose morphine treatment may cause increased cardiovascular morbidity in human subjects receiving higher therapeutic dose of morphine.

CONTENTS
Abstract in Chinese 1
Abstract in English 3
Chapter 1
Background
1-1. Pharmacology of morphine 5
1-2. Side Effects 7
1-3. Morphine Addiction/overuse 7
1-4. Effects of High-Dose Morphine 8
1-5. Research Hypotheses 9
1-5-1. Theme 1. Effects of high-dose morphine on vascular endothelial function. 9
1-5-2. Theme 2. Effects of high-dose morphine on angiogenesis and excisional wound closure. 9


Chapter 2
Theme 1: High-Dose Morphine Impairs Vascular Endothelial Function by Increased Production of Superoxide Anions
2-1. Introduction 10
2-2. Material and Methods
2-2-1. Animals Preparation and Drug Treatment 11
2-2-2. Measurement of Vascular Reactivity 12
2-2-3. Western Blot Analysis 13
2-2-4. Dihydroethidium Assay for Detection of Superoxide Anions 14
2-2-5. Cell Culture 14
2-2-6. Statistical Analysis 15
2-3. Results
2-3-1. Vascular Reactivity and Histology 15
2-3-2. Protein expression 16
2-3-3. Detection of superoxide anions in aortas 16
2-3-4. Effects of morphine on cultured HUVECs 16
2-4. Discussion 18



Chapter 3
Theme 2: Prolonged Use of High-Dose Morphine Impairs Angiogenesis and Mobilization of Endothelial Progenitor Cells in Mice.
3-1. Introduction 23
3-2. Material and Methods
3-2-1. Mouse model of wound healing 24
3-2-2. Physiological responses and blood analysis 26
3-2-3. Measurement of superoxide anions 26
3-2-4. Measurement of circulating endothelial progenitor cells 27
3-2-5. Murine angiogenesis assay 27
3-2-6. In vitro capillary tube formation assay 28
3-2-7. Statistical Analysis 29
3-3. Results
3-3-1. Effects of morphine on physiological responses and blood analysis 29
3-3-2. Effects of morphine on healing of excisional wound 29
3-3-3. Effects of morphine on superoxide production in wound and circulating MNCs 30
3-3-4. Effects of morphine on numbers of circulating EPCs 30
3-3-5. Effects of morphine on angiogenesis 30
3-4. Discussion 31


Figure 37
Table 49
References 51

References

(1) Trescot AM, Datta S, Lee M, Hansen H. Opioid pharmacology. Pain Physician 2008; 2 (suppl):S133-S153.
(2) Sawynok J. The therapeutic use of heroin: a review of the pharmacological literature. Can J Physiol Pharmacol 1986; 64:1-11.
(3) Umans JG, Inturrisi CE. Heroin: analgesia, toxicity and disposition in the mouse. Eur J Pharmacol 1982; 85:317-323.
(4) Robins LN. The Nathan B. Eddy Lecture: challenging conventional wisdom about drug abuse. NIDA Res Monogr 1994; 1994:30-45.
(5) Mather LE. 1994 John J. Bonica Lecture. The clinical effects of morphine pharmacology. Reg Anesth 1995; 20:263-282.
(6) Glare PA, Walsh TD. Clinical pharmacokinetics of morphine. Ther Drug Monit 1991; 13:1-23.
(7) Osborne R, Joel S, Trew D, Slevin M. Morphine and metabolite behavior after different routes of morphine administration: demonstration of the importance of the active metabolite morphine-6-glucuronide. Clin Pharmacol Ther 1990; 47:12-19.
(8) Christrup LL. Morphine metabolites. Acta Anaesthesiol Scand 1997; 41:116-122.
(9) Loh HH, Smith AP. Molecular characterization of opioid receptors. Annu Rev Pharmacol Toxicol 1990; 30:123-147.
(10) Mansour A, Watson SJ, Akil H. Opioid receptors: Past, present and future. Trends Neurosci 1995; 18:69-70.
(11) Rang HP, Dale MM, Ritter JM, Moore PK. Analgesic drugs. In: Rang HP, editor. Pharmacology. New York: Churchill Livingstone, 2003: 562-584.
(12) Jin YH, Nishioka H, Wakabayashi K, Fujita T, Yonehara N. Effect of morphine on the release of excitatory amino acids in the rat hind instep: Pain is modulated by the interaction between the peripheral opioid and glutamate systems. Neuroscience 2006; 138:1329-1399.
(13) Lamotte C, Pert CB, Snyder SH. Opiate receptor binding in primate spinal cord distribution and changes after dorsal root section. Brain Res 1976; 112:407-412.
(14) Heinricher MM, Schouten JC, Jobst EE. Activation of brainstem N-methyl-D-aspartate receptors is required for the analgesic actions of morphine given systemically. Pain 2001; 92:129-138.
(15) Isom GE, Meldrum MJ. Morphine induced respiratory depression: involvement of norepinephrine and serotonin. Proc West Pharmacol Soc 1980; 23:89-91.
(16) Duthie DJ, Nimmo WS. Adverse effects of opioid analgesic drugs. Br J Anaesth 1987; 59:61-77.
(17) Withington DE, Patrick JA, Reynolds F. Histamine release by morphine and diamorphine in man. Anaestheisa 1993; 48:26-29.
(18) Yu SL, Liu CP, Loy K, Lin SL. Acute myocardial infarction after heroin injections. Jpn Heart J 2004; 45:1021-1028.
(19) Sztajzel J, Karpuz H, Rutishauser W. Heroin abuse and myocardial infarction. Int J Cardiol 1994; 47:180-182.
(20) Rojavin M, Szabo I, Bussiere JL, Rogers TJ, Adler MW, Eisenstein TK. Morphine treatment in vitro or in vivo decreases phagocytic functions of murine macrophages. Life Sci 1993; 53:997-1006.
(21) Szabo I, Rojavin M, Bussiere JL, Eisenstein TK, Adler MW, Rogers TJ. Suppression of peritoneal macrophage phagocytosis of Candida albicans by opioids. J Pharmacol Exp Ther 1993; 267:703-706.
(22) Rook JM, McCarson KE. Delay of cutaneous wound closure by morphine via local blockade of peripheral tachykinin release. Biochem Pharmacol 2007; 74:752-757.
(23) Bhat RS, Bhaskaran M, Mongia A, Hitosugi N, Singhal PC. Morphine-induced macrophage apoptosis: oxidative stress and strategies for modulation. J Leukoc Biol 2004; 75:1131-1138.
(24) Singhal PC, Pamarthi M, Shah R, Chandra P, Gibbons N. Morphine stimulates superoxide formation by glomerular mesangial cells. Inflammation 1994; 18:293-299.
(25) Patel J, Manjappa N, Bhat R, Mehrotra P, Bhaskaran M, Singhal PC. Role of oxidative stress and heme oxygenase activity in morphine-induced glomerular epithelial cell growth. Am J Physiol Renal Physiol 2003; 285:861-869.
(26) Zhang YT, Zheng QS, Pan J, Zheng RL. Oxidative damage of biomolecules in mouse liver induced by morphine and protected by antioxidants. Basic Clin Pharmacol Toxicol 2004; 95:53-58.
(27) Cai H, Griendling KK, Harrison DG. The vascular NADPH oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci 2003; 24:471-478.
(28) Ray R, Shah AM. NADPH oxidase and endothelial cell function. Clin Sci 2005; 109:217-226.
(29) Brandes RP, Kreuzer J. Vascular NADPH oxidases: molecular mechanisms of activation. Cardiovasc Res 2005; 65:16-27.
(30) Suda O, Smith L, d'Uscio LV, Peterson TE, Katusic ZS. In vivo expression of recombinant vascular endothelial growth factor in rabbit carotid artery increases production of superoxide anion. Arterioscler Thromb Vasc Biol 2005; 25:506-511.
(31) Tsai YC, Won SJ, Lin MT. Effects of morphine on immune response in rats with sciatic constriction injury. Pain 2000; 88:155-160.
(32) Wong CL, Bentley GA. The effects of cholinergic compounds on the development of morphine tolerance, dependence and increased naloxone potency in mice. Eur J Pharmacol 1980; 61:99-109.
(33) Girardot MN, Holloway FA. Chronic stress, aging and morphine analgesia: chronic stress affects the reactivity to morphine in young mature but not old rats. J Pharmacol Exp Ther 1985; 233:545-553.
(34) Carun S, Goktalay G, Millington WR. Glycyl-glutamine, an endogenous beta-endorphin-derived peptide, inhibits morphine-induced conditioned place preference, tolerance, dependence, and withdrawal. J Pharmacol Exp Ther 2005; 315:949-958.
(35) Lam CF, Peterson TE, Richardson D, Croatt AJ, d'Uscio LV, Nath KA, Katusic ZS. Increased blood flow causes coordinated upregulation of arterial eNOS and biosynthesis of tetrahydrobiopterin. Am J Physiol Heart Circ Physiol 2006; 290:786-793.
(36) Lam CF, Peterson TE, Croatt AJ, Nath K, Katusic ZS. Functional adaptation and remodeling of pulmonary artery in flow-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 2005; 289:2334-2341.
(37) Azumi H, Inoue N, Ohashi Y, Terashima M, Mori T, Fujita H, Awano K, Kobayashi K, Maeda K, Hata K, Shinke T, Kobayashi S, Hirata K, Kawashima S, Itabe H, Hayashi Y, Imajoh-Ohmi S, Itoh H, Yokoyama M. Superoxide generation in directional coronary atherectomy specimens of patients with angina pectoris: important role of NAD(P)H oxidase. Arterioscler Thromb Vasc Biol 2002; 22:1838-1844.
(38) Eligini S, Stella Barbieri S, Cavalca V, Camera M, Brambilla M, De Franceschi M et al. Diversity and similarity in signaling events leading to rapid Cox-2 induction by tumor necrosis factor-alpha and phorbol ester in human endothelial cells. Cardiovasc Res 2005; 65:683-693.
(39) De Iuliis GN, Wingate JK, Koppers AJ, McLaughlin EA, Aitken RJ. Definitive evidence for the nonmitochondrial production of superoxide anion by human spermatozoa. J Clin Endocrinol Metab 2006; 91:1968-1975.
(40) Mueller CFH, Laude K, McNally JS, Harrison DG. Redox mechanisms in blood vessels. Arterioscler Thromb Vasc Biol 2005; 25:274-278.
(41) Li JM, Shah AM. Mechanism of endothelial cell NADPH oxidase activation by angiotensin II: Role of the p47phox subunit. J Biol Chem 2003; 278:12094-12100.
(42) Torres M, Forman HJ. Nitric oxide, oxidative stress, and signal transduction. In: Ignarro LJ, editor. Nitric Oxide: Biology and Pathobiology. New York: Academic Press, 2000: 329-342.
(43) Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Phyiol Cell Physiol 1996; 271:1424-1437.
(44) Stefano GB, Cadet P, Fimiani C, Magazine HI. Morphine stimulates iNOS expression via a rebound from inhibition in human macrophages: nitric oxide involvement. Int J Immunopathol Pharmacol 2001; 14:129-138.
(45) Benamar K, Yondorf MZ, Kon D, Geller EB, Adler MW. Role of the nitric-oxide synthase isoforms during morphine-induced hyperthermia in rats. J Pharmacol Exp Ther 2003; 307:219-222.
(46) Stefano GB, Burrill JD, Labur S, Blake J, Cadet P. Regulation of various genes in human leukocytes acutely exposed to morphine: expression in microarray analysis. Med Sci Monit 2005; 11:35-42.
(47) van der Loo B, Labugger R, Skepper JN, Bachschmid M, Kilo J, Powell JM, Palacios-Callender M, Erusalimsky JD, Quaschning T, Malinski T, Gygi D, Ullrich V, Lüscher TF. Enhanced peroxynitrite formation is associated with vascular aging. J Exp Med 2000; 192:1731-1744.
(48) Bauersachs J, Bouloumie A, Fraccarollo D, Hu K, Busse R, Ertl G. Endothelial dysfunction in chronic mayocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production. Circulation 1999; 100:292-298.
(49) Gupta K, Kshirsagar S, Chang L, Schwartz R, Law PY, Yee D, Hebbel RP. Morphine stimulates angiogenesis by activating proangiogenic and survival-promoting signaling and promotes breast tumor growth. Cancer Res 2002; 62:4491-4498.
(50) Liu HC, Anday JK, House SD, Chang SL. Dual effects of morphine on permeability and apoptosis of vascular endothelial cells: morphine potentiates lipopolysaccharide-induced permeability and apoptosis of vascular endothelial cells. J Neuroimmunol 2004; 146:13-21.
(51) Lam CF, Liu YC, Tseng FL, Sung YH, Huang CC, Jiang MJ et al. High-dose morphine impairs vascular endothelial function by increased production of superoxide anions. Anesthesiology 2007 ;106:532-7.
(52) Lee PC, Salyapongse AN, Bragdon GA, Shears II LL, Watkins SC, Edington HDJ, Billiar TR. Impaired wound healing and angiogenesis in eNOS-deficient mice. Am J Physiol Heart Circ Physiol 1999; 277:1600-1608.
(53) Balasubramanian S, Ramakrishnan S, Charboneau R, Wang J, Barke RA, Roy S. Morphine sulfate inhibits hypoxia-induced vascular endothelial growth factor expression in endothelial cells and cardiac myocytes. J Mol Cell Cardiol 2001; 33:2179-2187.
(54) Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275:964-967.
(55) Hristov M, Weber C. Endothelial progenitor cells: characterization, pathophysiology and possible clinical relevance. J Cell Mol Med 2004; 8:498-508.
(56) Suh W, Kim KL, Kim JM, Shin IS, Lee YS, Lee JY, Jang HS, Lee JS, Byun J, Choi JH, Jeon ES, Kim DK. Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes/macrophages and neovascularization. Stem Cells 2005; 23:1571-1578.
(57) Pacifici R, Patrini G, Venier I, Parolaro D, Zuccaro P, Gori E. Effect of morphine and methadone acute treatment on immunological activity in mice: pharmacokinetic and pharmacodynamic correlates. J Pharmacol Exp Ther 1994; 269:1112-1116.
(58) Pacifici R, di Carlo S, Bacosi A, Pichini S, Zuccaro P. Pharmacokinetics and cytokine production in heroin and morphine-treated mice. Int J Immunopharmac 2000; 22:603-614.
(59) Miyamoto Y, Morita N, Nakamura N, Yamanishi T, Kishioka S, Yamamoto H. Effect of naloxone on the morphine concentration in the central nervous system and plasma in rats. Japan J Pharmacol 1993; 63:235240.
(60) Wassmann S, Stumpf M, Strehlow K, Schmid A, Schieffer B, Bohm M, Nickenig G . Interleukin-6 induces oxidative stress and endothelial dysfunction by overexpression of the angiotensin II type 1 receptor. Circ Res 2004; 94:534-541.
(61) Asahara T, Kawamoto A. Endothelial progenitor cells for postnatal vasculogenesis. Am J Phyiol Cell Physiol 2004; 287:572-579.
(62) Shi CS, Shi GY, Chang YS, Han HS, Kuo CH, Liu C, Huang HC, Chang YJ, Chen PS, Wu HL. Evidence of human thrombomodulin domain as a novel angiogenetic factor. Circulation 2005; 111:1627-1636.
(63) Miura S, Matsuo Y, Saku K. Transactivation of KDR/Flk-1 by the B2 receptor induces tube formation in human coronary endothelial cells. Hypertension 2003; 41:1118-1123.
(64) Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005; 366:1736-1743.
(65) Wlaschek M, Scharffetter-Kochanek K. Oxidative stress in chronic venous leg ulcers. Wound Rep Reg 2005; 13:425-461.
(66) Singer AJ, Clark RA. Cutaneous wound healing. New Engl J Med 1999; 341:738-746.
(67) Moseley R, Hilton JR, Waddington RJ, Harding KG, Stephens P, Thomas DW. Comparison of oxidative stress biomarker profiles between acute and chronic wound environments. Wound Rep Reg 2004; 12:419-429.
(68) Rojkind M, Dominguez-Rosales JA, Nieto N, Greenwel P. Role of hydrogen peroxide and oxidative stress in healing responses. Cell Mol Life Sci 2002; 59:1872-1891.
(69) Poonawala T, Levay-Young BK, Hebbel RP, Gupta K. Opioids heal ischemic wounds in the rat. Wound Rep Reg 2005; 13:165-174.
(70) Li J, Zhang YP, Kirsner RS. Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix. Microsc Res Tech 2003; 60:107-114.
(71) DaCosta ML, Regan MC, al Sader M, Leader M, Bouchier-Hayes D. Diphenylhydantoin sodium promotes early and marked angiogenesis and results in increased collagen deposition and tensile strength in healing wounds. Surgery 1998; 123:287-293.
(72) Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999; 5:434-438.
(73) Urbich C, Heeschen C, Mildner-Rihm C, Urbich C, Ihling C, Technau-Ihling K, Zeiher AM, Dimmeler S. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med 2003; 9:1370-1376.
(74) Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. Neovascularization of ischemic myocardium by human bone marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling an dimproves cardiac function. Nat Med 2001; 7:430-436.
(75) Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, Asahara T. Therapeutic Potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001; 103:634-637.
(76) He T, Smith L, Harrington S, Nath K, Caplice NM, Katusic ZS. Transplantation of circulating endothelial progenitor cells restores endothelial function of denuded rabbit carotid arteries. Stroke 2004; 35:2378-2384.
(77) Kaushal S, Amiel GE, Guleserian KJ, Shapira OM, Perry T, Sutherland FW, Rabkin E, Moran AM, Schoen FJ, Atala A, Soker S, Bischoff J, Mayer JE Jr. Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med 2000; 7:1035-1040.
(78) Assmus B, Schachlinger V, Teupe C, Britten M, Lehmann R, Dobert N, Grünwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, Zeiher AM. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002; 106:3009-3017.
(79) Perin EC, Dohmann HFR, Borojevic R, Silva SA, Sousa AL, Mesquita CT, Rossi MI, Carvalho AC, Dutra HS, Dohmann HJ, Silva GV, Belém L, Vivacqua R, Rangel FO, Esporcatte R, Geng YJ, Vaughn WK, Assad JA, Mesquita ET, Willerson JT. Transendocardial, autologous bone marrow cell transplantation for sever, chronic ischemic heart failure. Circulation 2003; 107:2294-2302.
(80) Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Klinge H, Schümichen C, Nienaber CA, Freund M, Steinhoff G. Autologous bone marrow transplantation for myocardial regeneration. Lancet 2003; 361:45-46.
(81) Alexander M, Daniel T, Chaudry IH, Schwacha MG. Opiate analgesics contribute to the development of post-injury immunosuppression. J Surg Res 2005; 129:161-168.
(82) Pasi A, Qu BX, Steiner R, Senn HJ, Bar W, Messiha FS. Angiogenesis: modulation with opioids. Gen Pharmacol 1991; 22:1077-1079.
(83) Roy S, Balasubramanian S, Wang J, Chandrashekhar Y, Charboneau R, Barke R. Morphine inhibits VEGF expression in myocardial ischemia. Surgery 2003; 134:336-344.
(84) Rojas A, Figueroa H, Re L, Morales MA. Oxidative stress at the vascular wall. Mechanistic and pharmacological aspects. Arch Med Res 2006; 37:436-448.
(85) Fadini GP, Miorin M, Facco M, Bonamico S, Baesso I, Grego F, Menegolo M, de Kreutzenberg SV, Tiengo A, Agostini C, Avogaro A. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol 2005; 45:1449-1457.