- Email: [email protected]
Improved limb perfusion and neoangiogenesis after intramuscular erythropoietin infusion in experimental model of limb ischemia
Letters to the Editor
195
Improved limb perfusion and neoangiogenesis after intramuscular erythropoietin infusion in experimental model of limb ischemia Dimitris Tousoulis ⁎,1, Georgia Vogiatzi 1, Alexandros Briasoulis, Aggeliki Valatsou, Polyxeni Nikolopoulou, Konstantinos Papaxoinis, Alkistis Pantopoulou, Nikolaos Papageorgiou, Charalambos Antoniades, Despina Perrea, Christodoulos Stefanadis 1st Cardiology Unit, Hippokration Hospital, Athens University Medical School, Greece
a r t i c l e
i n f o
Article history: Received 23 May 2012 Received in revised form 26 June 2012 Accepted 21 August 2012 Available online 6 September 2012 Keywords: Atherosclerosis Angiogenesis Erythropoietin Ischemia
To the Editor Erythropoietin (EPO) is a hematopoietic cytokine which also enhances the mobilization of endothelial progenitor cells (EPCs) [1] and exerts a protective effect on endothelial cells in several vascular injury models [2]. In addition, neovascularization plays a role in diseases involving ischemia, including tumors, atherosclerotic plaques, and ischemic heart disease [3]. The angiopoietin (Ang)/Tie system has been reported to be critically involved in disease progression through the activation of signaling pathways that control angiogenic remodeling [4]. Although Ang-1 and Ang-2 share similar binding affinities for the Tie2 receptor (the tyrosine kinase with immunoglobulin and epidermal growth factor [EGF] homology domain 2 receptor), they have opposing effects on receptor activation. Ang-1 induces receptor phosphorylation and contributes to blood vessel stabilization by the recruitment of periendothelial cells [5]. Therefore, we sought to investigate the effects of erythropoietin administration on perfusion, neovascularization and angiogenic factors' expression in an experimental model of limb ischemia. More specifically, we aimed to assess its effects on blood flow and vascular endothelial growth factor (VEGF), Ang-2/Tie-2 expression in the ischemic limb. Mice were anesthetized with 100 mg/kg body weight ketamine hydrochloride IM (intramuscular) (Ketanest, Pharmacia). A midline incision was made in the left limb, permitting dissection to expose the femoral artery in the upper part of the left limb. The artery was ligated both proximally and distally using 5–0 silk suture and then excised, while all visible arterial branches between the proximal and distal ligations stay intact. Then, the incision was closed using 3–0 suture. All mice were kept at constant temperature (25 ± 1 °C) and humidity (60 ± 5%) with free access to normal chow and water throughout the experimental periods. The animals were sacrificed with cervical dislocation after they were anesthetized with inhaled halothane. The study was performed in accordance with the guidelines for animal experiments of Athens University School of Medicine
⁎ Corresponding author at: Athens University Medical School, Hippokration Hospital, Vasilissis Sofias 114, 115 28, Athens, Greece. Tel.: +30 2107782446; fax: +30 2107485039. E-mail address: [email protected] (D. Tousoulis). 1 These authors equally contributed.
and conformed to the Directive 2010/63/EU of the European Parliament. The mice were divided into four groups: 1, control group with hind limb ischemia with saline injection (0,2 ml, for 5 days); 2, EPO group with hind limb ischemia and subsequent treatment with EPO (400 IU/kg in 0,3 ml solution, for 5 days). To determine whether EPO facilitates restoration of blood vessel regeneration by injected BMCs, blood flow to the right and left hind limbs was assessed by scanning the lower abdomen and limbs of the mice with a scanning laser Doppler (Perimed, Sweden). On day 28 after EPO injection, the ischemic muscles (quadriceps, adductors) were dissected, postfixed, embedded in optimal cutting temperature compound (OCT) (Sakura Finetek Europe, Zoetermedium) and frozen at −20 °C. The immunofluorescence was performed on 50-μm-thick frozen sections using CD31 (PECAM-1) mouse anti-human monoclonal antibody and fluorescein isothiocyanate (FITC) labeled goat anti-mouse antibody. The glass slides were fixed in acetone at 20 °C for 20 min and rinsed in PBS. The CD31 antibody, diluted 1:100, was applied to the slides which were incubated at room temperature for 2 h. The slides were rinsed three times in PBS and then incubated with the FITCconjugated antibody in 1:50 dilution for 1 h at room temperature in the dark. The slides were then rinsed in PBS and mounted under a cover slip with the mounting medium. The slides were kept at 4 °C in the dark until scanning with laser microscopy. The fluorescent images were obtained with a Bio-Rad MRC-600 co focal laser scanning imaging system supplied with an argon ion laser source with a maximum emitting power of 25 mW and coupled to a Nikon Optiphot microscope under × 40 objective. The integrated excitation and emission filters (488 nm and 515 nm, respectively) were utilized for FITC (excitation peak 490 nm, emission maximum 525 nm). For each optical field, a z-series was collected. The confocal images were averaged 10 times by the Kalmann filter to reduce noise effects. A composite image was finally created consisting of a stack of 60 consecutive co focal images with a 0.35 μm step. The total area of the pixels labeled by the CD31 antibody on each optical field, detected above the threshold, was quantified automatically by the software. RTPCR was performed to analyze the expression of the mRNA of several angiogenic factors. Reverse transcription proceeded with 1 ml of total RNA using iScript cDNA Synthesis kit (Bio-Rad Laboratories). The primer sequences of the amplified product were as follows: VEGF: 5′CAG CTA TTG CCG TCC GAT TGA GA-3, ANG-1: 5′-CTG ATG GAC TGG GAA GGG AAC C-3′, ANG-2: 5′-GAA GGA CTG GGA AGG CAA CGA-3′, Tie-2: 5′-GAT TTT GGA TTG TCC CGA GGT CAA-3′, GAPDH: 5′-AAC TTT GTG AAG CTC ATT TCC TGG TAT-3′. Quantitative real-time RT-PCR was carried out with iCycler iQ (Bio-Rad Laboratories). The level of transcripts was shown as a relative expression level using GAPDH transcripts as a standard. The primer sets used in quantitative realtime RT-PCR for VEGF, Tie-2, Ang-1 and Ang-2 are available upon request. All studied variables are presented as percentages or mean plus/ minus standard error of mean (SEM), while medians and ranges were also calculated where this was required. Continuous variables were tested for normal distribution by Kolmogorov–Smirnov test. Differences between normally distributed variables were tested by one-way ANOVA followed by Bonferroni, Scheffe, and Tukey post hoc corrections, whereas differences between not normally distributed data
196
Letters to the Editor
Fig. 1. a, b, c. Ischemic/non ischemic ratio was significantly increased in ischemic limbs of EPO-treated mice versus control mice at 7 days and was maintained at 28 days compared to day 0. Capillary density was decreased after limb ischemia and was significantly higher in the EPO-treated groups compared to controls on day 28. Expression of Ang-1, Ang-2, VEGF, Tie-2 in the in the ischemic limbs of EPO-treated mice versus control mice (* indicates statistical significance p b 0.05).
Letters to the Editor Table 1 Flow values within each limb of interest in control and erythropoietin-treated mice. Limbs
Groups
N
Mean flow
Standard deviation (SD)
p Value
Baseline-ischemic
Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin
12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12
89.15 85.4 125.32 153.06 121.54 147.11 144.34 151.6 145.63 153.78 147.94 152.67 0.62 0.56 0.86 0.99 0.82 0.96
13.7 14.75 12.9 19.7 16.75 18.86 9.21 17.8 11.77 15.18 7.88 9.65 0.09 0.07 0.07 0.09 0.1 0.08
0.506
7 days-ischemic 28 days-ischemic Baseline-nonischemic 7 days-nonischemic 28 days-nonischemic Ischemic/nonischemic ratio baseline Ischemic/nonischemic ratio 7 days Ischemic/nonischemic ratio 28 days
0.001 0.002 0.223 0.156 0.203 0.088 0.001 0.001
Values are expressed as mean ± standard deviation (SD).
were tested by the nonparametric Kruskal–Wallis. All reported p values are based on two-sided tests and compared to a significance level of 5%. SPSS version 18.0 (SPSS, Chicago, IL) software was used for all the statistical calculations. Hind-limb ischemia resulted in decreased blood flow ratio (ischemic/ non-ischemic limb), as assessed by laser Doppler scanning from 0.97± 0.06 to 0.47±0.1 immediately after surgery. Ischemic/non ischemic ratio was significantly increased in ischemic limbs of EPO-treated mice versus control mice at 7 days (p=0.001 vs. control for EPO), which was maintained at 28 days (p=0.001 vs. control) (Fig.1a). In the control group blood flow was significantly increased on day 7 and day 28 compared to day 0; however in the EPO-treated group, blood flow was significantly higher than controls at every time evaluated (p=0.001). The mean blood flow of the ischemic and nonischemic limbs at baseline, 7 days and 28 days after limb ischemia are presented on Table 1. Furthermore, capillary density was significantly higher in the EPO-treated groups compared to controls on day 28 (pb 0.05) (Fig. 1b). There was no
0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2012.08.008
197
significant difference in the expression of Ang-1 in the ischemic limb between the EPO treated group (1.33±0.84 relative units [RLU]) and control group (3.88±1.76 and p=NS). However, the ischemic limb expressed significantly lower Ang-2 (9.1±1.5 RLU) in the control group compared to the ischemic limb (11.6±1.5 RLU, p=0.004) in the control animals (Fig. 1c). In addition, EPO induced a significant elevation of VEGF expression in the ischemic limb (5.16±3.2 RLU) compared to the nonischemic limb (1.03±0.86 RLU, p=0.003). Importantly, erythropoietin up-regulated the expression of Tie-2 in the ischemic limb (6.02±3.6RLU) compared to the control group (1.84±0.72RLU, p=0.003). No significant change of the Ang/Tie-2 and VEGF expression was observed in the nonischemic limbs. Based on our findings, we have shown that EPO enhanced angiogenesis seen during ischemia in the hind-limb model and that sustained Tie-2/Ang-2 upregulation in the ischemic muscle after EPO administration was critical for this enhancement. More specifically, we have shown that a 5-day course of EPO enhanced the recovery from ischemia via up-regulation of angiogenesis. At day 7 and day 28 after the induction of ischemia, blood flow was significantly improved in the EPO group. Capillary density was also increased at day 28, implying enhancement of revascularization in the EPO treated group. We also observed a remarkable increase of VEGF, Ang-2, and Tie-2 expression with a concomitant increase of capillary density and blood perfusion with the administration of EPO and our findings suggest that Ang-2 up-regulation promotes revascularization within the affected muscle. Therefore, elucidating the role of Ang2 in neovascularization could be important in understanding ischemic diseases. In addition, administration of EPO may be useful for supporting revascularization of ischemic tissues. The authors of this article have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Heeschen C, Aicher A, Lehmann R, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 2003;102:1340–6. [2] Heil M, Schaper W. Influence of mechanical, cellular, and molecular factors on collateral artery growth. Circ Res 2004;95:449–58. [3] Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653–60. [4] Sato TN, Tozawa Y, Deutsch U, et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 1995;376:70–4. [5] Davis S, Aldrich TH, Jones PF, et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 1996;87:1161–9.
195
Improved limb perfusion and neoangiogenesis after intramuscular erythropoietin infusion in experimental model of limb ischemia Dimitris Tousoulis ⁎,1, Georgia Vogiatzi 1, Alexandros Briasoulis, Aggeliki Valatsou, Polyxeni Nikolopoulou, Konstantinos Papaxoinis, Alkistis Pantopoulou, Nikolaos Papageorgiou, Charalambos Antoniades, Despina Perrea, Christodoulos Stefanadis 1st Cardiology Unit, Hippokration Hospital, Athens University Medical School, Greece
a r t i c l e
i n f o
Article history: Received 23 May 2012 Received in revised form 26 June 2012 Accepted 21 August 2012 Available online 6 September 2012 Keywords: Atherosclerosis Angiogenesis Erythropoietin Ischemia
To the Editor Erythropoietin (EPO) is a hematopoietic cytokine which also enhances the mobilization of endothelial progenitor cells (EPCs) [1] and exerts a protective effect on endothelial cells in several vascular injury models [2]. In addition, neovascularization plays a role in diseases involving ischemia, including tumors, atherosclerotic plaques, and ischemic heart disease [3]. The angiopoietin (Ang)/Tie system has been reported to be critically involved in disease progression through the activation of signaling pathways that control angiogenic remodeling [4]. Although Ang-1 and Ang-2 share similar binding affinities for the Tie2 receptor (the tyrosine kinase with immunoglobulin and epidermal growth factor [EGF] homology domain 2 receptor), they have opposing effects on receptor activation. Ang-1 induces receptor phosphorylation and contributes to blood vessel stabilization by the recruitment of periendothelial cells [5]. Therefore, we sought to investigate the effects of erythropoietin administration on perfusion, neovascularization and angiogenic factors' expression in an experimental model of limb ischemia. More specifically, we aimed to assess its effects on blood flow and vascular endothelial growth factor (VEGF), Ang-2/Tie-2 expression in the ischemic limb. Mice were anesthetized with 100 mg/kg body weight ketamine hydrochloride IM (intramuscular) (Ketanest, Pharmacia). A midline incision was made in the left limb, permitting dissection to expose the femoral artery in the upper part of the left limb. The artery was ligated both proximally and distally using 5–0 silk suture and then excised, while all visible arterial branches between the proximal and distal ligations stay intact. Then, the incision was closed using 3–0 suture. All mice were kept at constant temperature (25 ± 1 °C) and humidity (60 ± 5%) with free access to normal chow and water throughout the experimental periods. The animals were sacrificed with cervical dislocation after they were anesthetized with inhaled halothane. The study was performed in accordance with the guidelines for animal experiments of Athens University School of Medicine
⁎ Corresponding author at: Athens University Medical School, Hippokration Hospital, Vasilissis Sofias 114, 115 28, Athens, Greece. Tel.: +30 2107782446; fax: +30 2107485039. E-mail address: [email protected] (D. Tousoulis). 1 These authors equally contributed.
and conformed to the Directive 2010/63/EU of the European Parliament. The mice were divided into four groups: 1, control group with hind limb ischemia with saline injection (0,2 ml, for 5 days); 2, EPO group with hind limb ischemia and subsequent treatment with EPO (400 IU/kg in 0,3 ml solution, for 5 days). To determine whether EPO facilitates restoration of blood vessel regeneration by injected BMCs, blood flow to the right and left hind limbs was assessed by scanning the lower abdomen and limbs of the mice with a scanning laser Doppler (Perimed, Sweden). On day 28 after EPO injection, the ischemic muscles (quadriceps, adductors) were dissected, postfixed, embedded in optimal cutting temperature compound (OCT) (Sakura Finetek Europe, Zoetermedium) and frozen at −20 °C. The immunofluorescence was performed on 50-μm-thick frozen sections using CD31 (PECAM-1) mouse anti-human monoclonal antibody and fluorescein isothiocyanate (FITC) labeled goat anti-mouse antibody. The glass slides were fixed in acetone at 20 °C for 20 min and rinsed in PBS. The CD31 antibody, diluted 1:100, was applied to the slides which were incubated at room temperature for 2 h. The slides were rinsed three times in PBS and then incubated with the FITCconjugated antibody in 1:50 dilution for 1 h at room temperature in the dark. The slides were then rinsed in PBS and mounted under a cover slip with the mounting medium. The slides were kept at 4 °C in the dark until scanning with laser microscopy. The fluorescent images were obtained with a Bio-Rad MRC-600 co focal laser scanning imaging system supplied with an argon ion laser source with a maximum emitting power of 25 mW and coupled to a Nikon Optiphot microscope under × 40 objective. The integrated excitation and emission filters (488 nm and 515 nm, respectively) were utilized for FITC (excitation peak 490 nm, emission maximum 525 nm). For each optical field, a z-series was collected. The confocal images were averaged 10 times by the Kalmann filter to reduce noise effects. A composite image was finally created consisting of a stack of 60 consecutive co focal images with a 0.35 μm step. The total area of the pixels labeled by the CD31 antibody on each optical field, detected above the threshold, was quantified automatically by the software. RTPCR was performed to analyze the expression of the mRNA of several angiogenic factors. Reverse transcription proceeded with 1 ml of total RNA using iScript cDNA Synthesis kit (Bio-Rad Laboratories). The primer sequences of the amplified product were as follows: VEGF: 5′CAG CTA TTG CCG TCC GAT TGA GA-3, ANG-1: 5′-CTG ATG GAC TGG GAA GGG AAC C-3′, ANG-2: 5′-GAA GGA CTG GGA AGG CAA CGA-3′, Tie-2: 5′-GAT TTT GGA TTG TCC CGA GGT CAA-3′, GAPDH: 5′-AAC TTT GTG AAG CTC ATT TCC TGG TAT-3′. Quantitative real-time RT-PCR was carried out with iCycler iQ (Bio-Rad Laboratories). The level of transcripts was shown as a relative expression level using GAPDH transcripts as a standard. The primer sets used in quantitative realtime RT-PCR for VEGF, Tie-2, Ang-1 and Ang-2 are available upon request. All studied variables are presented as percentages or mean plus/ minus standard error of mean (SEM), while medians and ranges were also calculated where this was required. Continuous variables were tested for normal distribution by Kolmogorov–Smirnov test. Differences between normally distributed variables were tested by one-way ANOVA followed by Bonferroni, Scheffe, and Tukey post hoc corrections, whereas differences between not normally distributed data
196
Letters to the Editor
Fig. 1. a, b, c. Ischemic/non ischemic ratio was significantly increased in ischemic limbs of EPO-treated mice versus control mice at 7 days and was maintained at 28 days compared to day 0. Capillary density was decreased after limb ischemia and was significantly higher in the EPO-treated groups compared to controls on day 28. Expression of Ang-1, Ang-2, VEGF, Tie-2 in the in the ischemic limbs of EPO-treated mice versus control mice (* indicates statistical significance p b 0.05).
Letters to the Editor Table 1 Flow values within each limb of interest in control and erythropoietin-treated mice. Limbs
Groups
N
Mean flow
Standard deviation (SD)
p Value
Baseline-ischemic
Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin Control Erythropoietin
12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12
89.15 85.4 125.32 153.06 121.54 147.11 144.34 151.6 145.63 153.78 147.94 152.67 0.62 0.56 0.86 0.99 0.82 0.96
13.7 14.75 12.9 19.7 16.75 18.86 9.21 17.8 11.77 15.18 7.88 9.65 0.09 0.07 0.07 0.09 0.1 0.08
0.506
7 days-ischemic 28 days-ischemic Baseline-nonischemic 7 days-nonischemic 28 days-nonischemic Ischemic/nonischemic ratio baseline Ischemic/nonischemic ratio 7 days Ischemic/nonischemic ratio 28 days
0.001 0.002 0.223 0.156 0.203 0.088 0.001 0.001
Values are expressed as mean ± standard deviation (SD).
were tested by the nonparametric Kruskal–Wallis. All reported p values are based on two-sided tests and compared to a significance level of 5%. SPSS version 18.0 (SPSS, Chicago, IL) software was used for all the statistical calculations. Hind-limb ischemia resulted in decreased blood flow ratio (ischemic/ non-ischemic limb), as assessed by laser Doppler scanning from 0.97± 0.06 to 0.47±0.1 immediately after surgery. Ischemic/non ischemic ratio was significantly increased in ischemic limbs of EPO-treated mice versus control mice at 7 days (p=0.001 vs. control for EPO), which was maintained at 28 days (p=0.001 vs. control) (Fig.1a). In the control group blood flow was significantly increased on day 7 and day 28 compared to day 0; however in the EPO-treated group, blood flow was significantly higher than controls at every time evaluated (p=0.001). The mean blood flow of the ischemic and nonischemic limbs at baseline, 7 days and 28 days after limb ischemia are presented on Table 1. Furthermore, capillary density was significantly higher in the EPO-treated groups compared to controls on day 28 (pb 0.05) (Fig. 1b). There was no
0167-5273/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2012.08.008
197
significant difference in the expression of Ang-1 in the ischemic limb between the EPO treated group (1.33±0.84 relative units [RLU]) and control group (3.88±1.76 and p=NS). However, the ischemic limb expressed significantly lower Ang-2 (9.1±1.5 RLU) in the control group compared to the ischemic limb (11.6±1.5 RLU, p=0.004) in the control animals (Fig. 1c). In addition, EPO induced a significant elevation of VEGF expression in the ischemic limb (5.16±3.2 RLU) compared to the nonischemic limb (1.03±0.86 RLU, p=0.003). Importantly, erythropoietin up-regulated the expression of Tie-2 in the ischemic limb (6.02±3.6RLU) compared to the control group (1.84±0.72RLU, p=0.003). No significant change of the Ang/Tie-2 and VEGF expression was observed in the nonischemic limbs. Based on our findings, we have shown that EPO enhanced angiogenesis seen during ischemia in the hind-limb model and that sustained Tie-2/Ang-2 upregulation in the ischemic muscle after EPO administration was critical for this enhancement. More specifically, we have shown that a 5-day course of EPO enhanced the recovery from ischemia via up-regulation of angiogenesis. At day 7 and day 28 after the induction of ischemia, blood flow was significantly improved in the EPO group. Capillary density was also increased at day 28, implying enhancement of revascularization in the EPO treated group. We also observed a remarkable increase of VEGF, Ang-2, and Tie-2 expression with a concomitant increase of capillary density and blood perfusion with the administration of EPO and our findings suggest that Ang-2 up-regulation promotes revascularization within the affected muscle. Therefore, elucidating the role of Ang2 in neovascularization could be important in understanding ischemic diseases. In addition, administration of EPO may be useful for supporting revascularization of ischemic tissues. The authors of this article have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Heeschen C, Aicher A, Lehmann R, et al. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 2003;102:1340–6. [2] Heil M, Schaper W. Influence of mechanical, cellular, and molecular factors on collateral artery growth. Circ Res 2004;95:449–58. [3] Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653–60. [4] Sato TN, Tozawa Y, Deutsch U, et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 1995;376:70–4. [5] Davis S, Aldrich TH, Jones PF, et al. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 1996;87:1161–9.