Transgenic Res DOI 10.1007/s11248-014-9829-5
BRIEF COMMUNICATION
Impaired erythrocyte deformability in transgenic HO-1G143H mutant mice Gan Chen • Yujing Yin • Bo Wang • Penglong Li Qingjun Liu • Guoxing You • Jingxiang Zhao • Sha Xia • Lian Zhao • Hong Zhou
•
Received: 28 November 2013 / Accepted: 12 August 2014 Ó Springer International Publishing Switzerland 2014
Abstract To investigate the potential effects of variation of HO-1 activity on hemorheology, this study compared the hemorheological properties between transgenic HO-1G143H mutant mice and wild-type (WT) control mice. Fresh blood samples were obtained from mice via the ocular venous sinus. The whole blood viscosity was measured using a cone–plate viscometer. Erythrocyte deformability and aggregation was measured using ektacytometry. The elongation index was significantly reduced in the HO1G143H mutant mice compared to the WT mice at the shear rates of 600, 800, and 1,000 s-1. The integrated elongation index was decreased in the HO-1G143H mutant mice compared to the WT mice. There was no statistically significant difference between the HO1G143H mutant mice and the WT mice in terms of whole blood viscosity, aggregation index, amplitude of aggregation, and aggregation half time. The present
Gan Chen and Yujing Yin have contributed equally to this work. G. Chen Y. Yin B. Wang P. Li Q. Liu G. You J. Zhao S. Xia L. Zhao (&) H. Zhou (&) Institute of Transfusion Medicine, Academy of Military Medical Sciences, No. 27th Taiping Road, HaiDian, Beijing, China e-mail: [email protected] H. Zhou e-mail: [email protected]
study demonstrated that a reduction in HO-1 activity results in an impaired erythrocyte deformability. Although the mechanism underlying this effect remains unclear, our study brings to light the participation of HO-1 in the variations of hemorheology. Keywords Heme oxygenase-1 Hemorheology Erythrocyte deformability Erythrocyte aggregation Whole blood viscosity
Introduction Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme catabolism (Gozzelino et al. 2010). Accumulating evidence has suggested that HO-1 expression is induced by a large number of oxidative stress stimuli and has potent cytoprotective and anti-inflammatory functions (Immenschuh and Schroder 2006). However, HO-1 activity varies in response to given stimuli in humans. It has been shown that the polymorphism in the HO-1 promoter modulates the transcriptional activity of the HO-1 gene (Exner et al. 2004). Individuals with attenuated HO-1 activity were associated with a high risk for hypertension, diabetes mellitus, and cardiovascular disease (CAD) (Kaneda et al. 2002; Chen et al. 2012b; Bao et al. 2010). The only known case of human HO-1 deficiency exhibited marked hemolytic anemia, endothelial injury, and
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oxidative stress (Kawashima et al. 2002; Yachie et al. 1999). Impaired hemorheology has been demonstrated in many pathological conditions and has been proven to play an important role in the pathogenesis and development of some diseases, such as cardiovascular disease, hypertension, diabetes mellitus, and hemolytic anemia (Keymel et al. 2011; Chen et al. 2012a; Allard et al. 1978). Impaired erythrocyte deformability compromises the ability of erythrocytes to pass through capillaries, reduces tissue perfusion, and impairs tissue oxygen supply (Hach et al. 2008; Parthasarathi and Lipowsky 1999), all of which contribute to endothelial injury and microcirculatory disturbances under these pathological conditions (Rogausch 1973; MacRury and Lowe 1990). However, to our knowledge there has been no study published focusing on the effects of the variation of HO-1 activity on hemorheology. We hypothesized that the inherent variation of HO-1 activity in humans impacts the hemorheological parameters. Consequently, the aim of this study was to examine the influence of HO-1 activity on the hemorheology by comparing the hemorheological parameters between transgenic HO-1G143H mutant mice (a mice model mimicking HO-1 deficiency by HO-1G143H mutant overexpression) and wild-type (WT) control mice. The hemorheological parameters include blood viscosity, erythrocyte aggregation, and erythrocyte deformability.
Materials and methods Animals This study was approved by the ethics committee of the Institute of Transfusion Medicine, Academy of Military Medical Sciences. All efforts were made to minimize the number of animals used and their suffering. Thirty HO-1G143H mutant and WT mice (10–12 weeks) were included in the study. The transgenic HO-1G143H mutant mice were produced as described previously (Liu et al. 2013). The offspring was identified by reverse transcriptionpolymerase chain reaction, and the negative mice were used as the controls.
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Blood sampling Fresh blood samples were obtained from mice via the ocular venous sinus using heparin-treated tubes after anaesthetization with ethyl ether. Hemorheological evaluation The whole blood viscosity was measured at shear rates of 10, 50, and 150 s-1 using a cone–plate viscometer (LBY-N6B, Precil Company, Beijing, China). Erythrocyte deformability was determined using an ektacytometer based on laser diffraction (LBY-BX, Precil Company, Beijing, China) at shear rates of 100, 400, 600, 800, and 1,000 s-1. For this purpose, a 20-ll blood sample was mixed with 1 mL viscous solution of polyvinylpyrrolidone (PVP, MW = 30 kDa) in an isotonic phosphate buffer base (pH 7.4). Erythrocyte aggregation was also measured using an ektacytometer at 37 °C. The measurement was based on the change in back-scattered light on abrupt cessation of the erythrocyte suspension (Hardeman et al. 2001). After mixing 420 ll of blood with 110 ll of PVP buffer, a 500-ll mixture was used to measure the aggregation parameters, including aggregation index (AI), aggregation half time (t1/2), and aggregation amplitude (AMP). Statistical analysis Data are expressed as the mean ± standard deviation (SD). Statistical comparisons between groups were evaluated using ANOVA followed by the Student– Newman–Keuls test when the normality and homogeneity of variance assumptions were satisfied; otherwise, ANOVA followed by Student–Newman– Keuls multiple range text were applied. P \ 0.05 was considered significant.
Results Whole blood viscosity The whole blood viscosity measurements are presented in Fig. 1. There were no significantly differences between the two groups at the various shear rates.
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Erythrocyte aggregation The AI, AMP, and t1/2 values are shown in Fig. 3. Although transgenic HO-1G143H mutant mice displayed a trend of increased AI and AMP and decreased t1/2, there was no statistically significant difference between the two groups for AI, AMP, and t 1/2.
Discussion
Fig. 1 The whole blood viscosity in HO-1G143H mutant mice and WT mice. Data are plotted as the mean ± SD (n = 5)
Erythrocyte deformability The erythrocyte deformability measurements are presented in Fig. 2. The elongation index (EI) or integrated EI reflects erythrocyte deformability. EI was significantly reduced in the HO-1G143H mutant mice compared to the WT mice at the shear rates of 600, 800, and 1,000 s-1 (P \ 0.05). In addition, the integrated EI was decreased in the HO-1G143H mutant mice compared to WT mice (P \ 0.05).
The present study demonstrated that a reduction in HO-1 activity in HO-1G143H mutant mice results in impaired erythrocyte deformability. There is no significant difference with respect to erythrocyte aggregation and whole blood viscosity between the HO1G143H mutant mice and the WT mice. Erythrocyte deformability is of fundamental importance for maintaining normal circulation. Reduced erythrocyte deformability contributes to impaired microcirculatory perfusion (Simchon et al. 1987), which results in hypoxia and endothelial dysfunction (Yang et al. 2007; Chen et al. 2012a). Furthermore, a slight decrease in erythrocyte deformability may reduce the rate of entry of erythrocytes into the capillaries and may subsequently impair cellular oxygen delivery and tissue oxygenation (Cicco and Pirrelli 1999). Erythrocyte deformability is regulated by multiple factors, such as oxidative stress and nitric oxide (NO).
Fig. 2 Impaired erythrocyte deformability in HO-1G143H mutant mice. a Erythrocyte deformability (EI) at several shear rates in mice. b Integrated EI in mice. Data are plotted as the mean ± SD (n = 5). #P \ 0.05 versus the WT mice
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Fig. 3 Comparison of erythrocyte aggregation in HO-1G143H mutant mice and WT mice. a AI in mice. b t1/2 in mice. c AMP in mice. Data are plotted as the mean ± SD (n = 5)
Oxidative stress has been reported to decrease RBC deformability in vitro and in vivo (Aydogan et al. 2008; Mohanty et al. 2013). In addition, nitric oxide (NO) is responsible for maintaining normal erythrocyte deformability (Bor-Kucukatay et al. 2003). Decreased NO levels may also contribute to impaired erythrocyte deformability. HO-1 has a potential protective function against oxidative stress (Soares and Bach 2009). HO-1 deficient mice show enhanced levels of oxidative damage (Poss and Tonegawa 1997; True et al. 2007). Furthermore, HO-1 and the metabolic products of heme can also increase the half-life of NO by reducing oxidative stress (Wu and Wang 2005). A previous study indicated that inhibition of HO-1 activity by SnPP results in decreased levels of NOx, which is a stable end product of NO (Ishikawa
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et al. 2001). Consequently, the impaired erythrocyte deformability in transgenic HO-1G143H mutant mice may result from the enhanced oxidative stress and the consequential reduction in generation and half-life of NO. On the other hand, the dysfunction of abnormal erythrocyte clearance system may also contribute to the impaired erythrocyte deformability in transgenic HO-1G143H mutant mice. It is well known that the spleen serves as the largest filter of blood by initiating immune responses and removing abnormal blood cells (Mebius and Kraal 2005). The structural and mechanical quality of the erythrocytes is ascertained by the mechanical constraint imposed by the meshwork in the red pulp, where old and abnormal erythrocytes that are less deformable are retained and eventually
Transgenic Res
removed by phagocytosis (Huang et al. 2014). However, splenic macrophages that phagocytose senescent erythrocytes die in HO-1-/- mice. The contents released from dying macrophages include nonmetabolized heme, which damages surrounding cells and causes fibrosis in red pulp areas of the spleen, thereby eliminating the normal setting in which recycling of erythrocytes usually occurs (Kovtunovych et al. 2010). Our previous study showed that the HO1G143H mice presented with anemia, enlarged spleen and tissue iron overload, which was similar to HO-1-/- mice (Zhou et al. 2011). So, the abnormal erythrocytes that are less deformable may no longer efficiently removed from the circulation in HO1G143H mice, which contributed to the impaired erythrocyte deformability that we observed. In the present study, there was no significant difference in erythrocyte aggregation, although the transgenic HO-1G143H mutant mice displayed a trend of increased erythrocyte aggregation compared with the WT mice. As mentioned above, HO-1 activity deficient mice showed enhanced levels of oxidative damage. The oxidative damage may accelerate erythrocyte aggregation (Baskurt et al. 1998). One possible explanation for the lack of a significant difference between the two models is that other factors such as erythrocyte properties simultaneously affect erythrocyte aggregation. For example, it has been shown that erythrocyte aggregation can be suppressed by decreased cell deformability (Cicha et al. 1999; Muralidharan et al. 1994; Jovtchev et al. 2000; Reinhart and Singh 1990). In addition, blood viscosity was not significantly different demonstrating that the decreased erythrocyte deformability in transgenic HO1G143H mutant mice had no significant effect on blood viscosity.
Conclusions The current experimental data indicate that a reduction in erythrocyte deformability in transgenic HO1G143H mutant mice. The possible mechanisms may associate with enhanced oxidative stress, decreased NO levels, and the dysfunction of abnormal erythrocyte clearance system in transgenic HO1G143H mutant mice. Our study has limitation in that the mechanism mentioned above were not verified by experimental data. Although the mechanism
underlying this effect remains unclear, our study brings to light the participation of HO-1 in the variations of hemorheology and the limitations will be addressed in continued studies. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 31271001) and the National High Technology Research and Development Program of China (No. 2012AA021902). Conflict of interest
None declared.
References Allard C, Mohandas N, Bessis M (1978) Red cell deformability changes in hemolytic anemias estimated by diffractometric methods (ektacytometry) preliminary results. In: Bessis M, Shohet S, Mohandas N (eds) Red Cell Rheology. Springer, Berlin, pp 209–221 Aydogan S, Yapislar H, Artis S, Aydogan B (2008) Impaired erythrocytes deformability in H2O2-induced oxidative stress: protective effect of L-carnosine. Clin Hemorheol Microcirc 39(1):93–98 Bao W, Song F, Li X, Rong S, Yang W, Wang D, Xu J, Fu J, Zhao Y, Liu L (2010) Association between heme oxygenase-1 gene promoter polymorphisms and type 2 diabetes mellitus: a HuGE review and meta-analysis. Am J Epidemiol 172(6):631–636 Baskurt OK, Temiz A, Meiselman HJ (1998) Effect of superoxide anions on red blood cell rheologic properties. Free Radic Biol Med 24(1):102–110 Bor-Kucukatay M, Wenby RB, Meiselman HJ, Baskurt OK (2003) Effects of nitric oxide on red blood cell deformability. Am J Physiol Heart Circ Physiol 284(5):1577–1584 Chen G, Zhao L, Liu Y, Liao F, Han D, Zhou H (2012a) Regulation of blood viscosity in disease prevention and treatment. Chin Sci Bull 57(16):1946–1952 Chen M, Zhou L, Ding H, Huang S, He M, Zhang X, Cheng L, Wang D, Hu FB, Wu T (2012b) Short (GT) (n) repeats in heme oxygenase-1 gene promoter are associated with lower risk of coronary heart disease in subjects with high levels of oxidative stress. Cell Stress Chaperones 17(3): 329–338 Cicco G, Pirrelli A (1999) Red blood cell (RBC) deformability, RBC agreeability and tissue oxygenation in hypertension. Clin Hemorheol Microcirc 21(3):169–177 Cicha I, Suzuki Y, Tateishi N, Maeda N (1999) Rheological changes in human red blood cells under oxidative stress. Pathophysiology 6(2):103–110 Exner M, Minar E, Wagner O, Schillinger M (2004) The role of heme oxygenase-1 promoter polymorphisms in human disease. Free Radic Biol Med 37(8):1097–1104 Gozzelino R, Jeney V, Soares MP (2010) Mechanisms of cell protection by heme oxygenase-1. Annu Rev Pharmacol Toxicol 50:323–354 Hach T, Forst T, Kunt T, Ekberg K, Pfutzner A, Wahren J (2008) C-peptide and its C-terminal fragments improve
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Transgenic Res erythrocyte deformability in type 1 diabetes patients. Exp Diabetes Res 2008:730594 Hardeman MR, Dobbe JGG, Ince C (2001) The laser-assisted optical rotational cell analyzer (LORCA) as red blood cell aggregometer. Clin Hemorheol Microcirc 25(1):1–11 Huang S, Amaladoss A, Liu M, Chen H, Zhang R, Preiser PR, Dao M, Han J (2014) In vivo splenic clearance correlates with in vitro deformability of red blood cells from Plasmodium yoelii-infected mice. Infect Immun 82(6):2532– 2541 Immenschuh S, Schroder H (2006) Heme oxygenase-1 and cardiovascular disease. Histol Histopathol 21(6):679–685 Ishikawa K, Sugawara D, Wang X-p, Suzuki K, Itabe H, Maruyama Y, Lusis AJ (2001) Heme oxygenase-1 inhibits atherosclerotic lesion formation in LDL-receptor knockout mice. Circ Res 88(5):506–512 Jovtchev S, Djenev I, Stoeff S, Stoylov S (2000) Role of electrical and mechanical properties of red blood cells for their aggregation. Colloids Surf Physicochem Eng Aspects 164(2–3):95–104 Kaneda H, Ohno M, Taguchi J, Togo M, Hashimoto H, Ogasawara K, Aizawa T, Ishizaka N, Nagai R (2002) Heme oxygenase-1 gene promoter polymorphism is associated with coronary artery disease in Japanese patients with coronary risk factors. Arterioscler Thromb Vasc Biol 22(10):1680–1685 Kawashima A, Oda Y, Yachie A, Koizumi S, Nakanishi I (2002) Heme oxygenase–1 deficiency: the first autopsy case. Hum Pathol 33(1):125–130 Keymel S, Heiss C, Kleinbongard P, Kelm M, Lauer T (2011) Impaired red blood cell deformability in patients with coronary artery disease and diabetes mellitus. Horm Metab Res 43(11):760–765 Kovtunovych G, Eckhaus MA, Ghosh MC, Ollivierre-Wilson H, Rouault TA (2010) Dysfunction of the heme recycling system in heme oxygenase 1-deficient mice: effects on macrophage viability and tissue iron distribution. Blood 116(26):6054–6062 Liu Q, Wang B, Yin Y, Chen G, Wang W, Gao X, Wang P, Zhou H (2013) Overexpressions of HO-1/HO-1G143H in C57/ B6 J mice affect melanoma B16F10 lung metastases rather than change the survival rate of mice-bearing tumours. Exp Biol Med 238(6):696–704 MacRury SM, Lowe GDO (1990) Blood rheology in diabetes mellitus. Diabet Med 7(4):285–291 Mebius RE, Kraal G (2005) Structure and function of the spleen. Nat Rev Immunol 5(8):606–616
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Mohanty JG, Nagababu E, Friedman JS, Rifkind JM (2013) SOD2 deficiency in hematopoietic cells in mice results in reduced red blood cell deformability and increased heme degradation. Exp Hematol 41(3):316–321 Muralidharan E, Tateishi N, Maeda N (1994) Simultaneous influence of erythrocyte deformability and macromolecules in the medium on erythrocyte aggregation: a kinetic study by a laser scattering technique. BBA—Biomembranes 1194(2):255–263 Parthasarathi K, Lipowsky HH (1999) Capillary recruitment in response to tissue hypoxia and its dependence on red blood cell deformability. Am J Physiol 277(6):H2145–H2157 Poss KD, Tonegawa S (1997) Heme oxygenase 1 is required for mammalian iron reutilization. Proc Natl Acad Sci USA 94(20):10919–10924 Reinhart WH, Singh A (1990) Erythrocyte aggregation: the roles of cell deformability and geometry. Eur J Clin Invest 20(4):458–462 Rogausch H (1973) Influence of erythrocyte deformability on circulation. Res Exp Med (Berl) 160(2):89–94 Simchon S, Jan KM, Chien S (1987) Influence of reduced red cell deformability on regional blood flow. Am J Physiol Heart Circ Physiol 253(4):H898–H903 Soares MP, Bach FH (2009) Heme oxygenase-1: from biology to therapeutic potential. Trends Mol Med 15(2):50–58 True AL, Olive M, Boehm M, San H, Westrick RJ, Raghavachari N, Xu X, Lynn EG, Sack MN, Munson PJ, Gladwin MT, Nabel EG (2007) Heme oxygenase-1 deficiency accelerates formation of arterial thrombosis through oxidative damage to the endothelium, which is rescued by inhaled carbon monoxide. Circ Res 101(9):893–901 Wu L, Wang R (2005) Carbon monoxide: endogenous production, physiological functions, and pharmacological applications. Pharmacol Rev 57(4):585–630 Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, Koizumi S (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Invest 103(1):129–135 Yang ZC, Xia K, Wang L, Jia SJ, Li D, Zhang Z, Deng S, Zhang XH, Deng HW, Li YJ (2007) Asymmetric dimethylarginine reduced erythrocyte deformability in streptozotocininduced diabetic rats. Microvasc Res 73(2):131–136 Zhou L, Liu Y, Zou C, Ma N, Hui Y, Lv G, Zhang H, Zhou H, Gao X (2011) The effect of the Gly139His, Gly143His, and Ser142His mouse heme oxygenase-1 mutants on the HO reaction in vivo and in vitro. Anat Rec 294(1):112–118
BRIEF COMMUNICATION
Impaired erythrocyte deformability in transgenic HO-1G143H mutant mice Gan Chen • Yujing Yin • Bo Wang • Penglong Li Qingjun Liu • Guoxing You • Jingxiang Zhao • Sha Xia • Lian Zhao • Hong Zhou
•
Received: 28 November 2013 / Accepted: 12 August 2014 Ó Springer International Publishing Switzerland 2014
Abstract To investigate the potential effects of variation of HO-1 activity on hemorheology, this study compared the hemorheological properties between transgenic HO-1G143H mutant mice and wild-type (WT) control mice. Fresh blood samples were obtained from mice via the ocular venous sinus. The whole blood viscosity was measured using a cone–plate viscometer. Erythrocyte deformability and aggregation was measured using ektacytometry. The elongation index was significantly reduced in the HO1G143H mutant mice compared to the WT mice at the shear rates of 600, 800, and 1,000 s-1. The integrated elongation index was decreased in the HO-1G143H mutant mice compared to the WT mice. There was no statistically significant difference between the HO1G143H mutant mice and the WT mice in terms of whole blood viscosity, aggregation index, amplitude of aggregation, and aggregation half time. The present
Gan Chen and Yujing Yin have contributed equally to this work. G. Chen Y. Yin B. Wang P. Li Q. Liu G. You J. Zhao S. Xia L. Zhao (&) H. Zhou (&) Institute of Transfusion Medicine, Academy of Military Medical Sciences, No. 27th Taiping Road, HaiDian, Beijing, China e-mail: [email protected] H. Zhou e-mail: [email protected]
study demonstrated that a reduction in HO-1 activity results in an impaired erythrocyte deformability. Although the mechanism underlying this effect remains unclear, our study brings to light the participation of HO-1 in the variations of hemorheology. Keywords Heme oxygenase-1 Hemorheology Erythrocyte deformability Erythrocyte aggregation Whole blood viscosity
Introduction Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme catabolism (Gozzelino et al. 2010). Accumulating evidence has suggested that HO-1 expression is induced by a large number of oxidative stress stimuli and has potent cytoprotective and anti-inflammatory functions (Immenschuh and Schroder 2006). However, HO-1 activity varies in response to given stimuli in humans. It has been shown that the polymorphism in the HO-1 promoter modulates the transcriptional activity of the HO-1 gene (Exner et al. 2004). Individuals with attenuated HO-1 activity were associated with a high risk for hypertension, diabetes mellitus, and cardiovascular disease (CAD) (Kaneda et al. 2002; Chen et al. 2012b; Bao et al. 2010). The only known case of human HO-1 deficiency exhibited marked hemolytic anemia, endothelial injury, and
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oxidative stress (Kawashima et al. 2002; Yachie et al. 1999). Impaired hemorheology has been demonstrated in many pathological conditions and has been proven to play an important role in the pathogenesis and development of some diseases, such as cardiovascular disease, hypertension, diabetes mellitus, and hemolytic anemia (Keymel et al. 2011; Chen et al. 2012a; Allard et al. 1978). Impaired erythrocyte deformability compromises the ability of erythrocytes to pass through capillaries, reduces tissue perfusion, and impairs tissue oxygen supply (Hach et al. 2008; Parthasarathi and Lipowsky 1999), all of which contribute to endothelial injury and microcirculatory disturbances under these pathological conditions (Rogausch 1973; MacRury and Lowe 1990). However, to our knowledge there has been no study published focusing on the effects of the variation of HO-1 activity on hemorheology. We hypothesized that the inherent variation of HO-1 activity in humans impacts the hemorheological parameters. Consequently, the aim of this study was to examine the influence of HO-1 activity on the hemorheology by comparing the hemorheological parameters between transgenic HO-1G143H mutant mice (a mice model mimicking HO-1 deficiency by HO-1G143H mutant overexpression) and wild-type (WT) control mice. The hemorheological parameters include blood viscosity, erythrocyte aggregation, and erythrocyte deformability.
Materials and methods Animals This study was approved by the ethics committee of the Institute of Transfusion Medicine, Academy of Military Medical Sciences. All efforts were made to minimize the number of animals used and their suffering. Thirty HO-1G143H mutant and WT mice (10–12 weeks) were included in the study. The transgenic HO-1G143H mutant mice were produced as described previously (Liu et al. 2013). The offspring was identified by reverse transcriptionpolymerase chain reaction, and the negative mice were used as the controls.
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Blood sampling Fresh blood samples were obtained from mice via the ocular venous sinus using heparin-treated tubes after anaesthetization with ethyl ether. Hemorheological evaluation The whole blood viscosity was measured at shear rates of 10, 50, and 150 s-1 using a cone–plate viscometer (LBY-N6B, Precil Company, Beijing, China). Erythrocyte deformability was determined using an ektacytometer based on laser diffraction (LBY-BX, Precil Company, Beijing, China) at shear rates of 100, 400, 600, 800, and 1,000 s-1. For this purpose, a 20-ll blood sample was mixed with 1 mL viscous solution of polyvinylpyrrolidone (PVP, MW = 30 kDa) in an isotonic phosphate buffer base (pH 7.4). Erythrocyte aggregation was also measured using an ektacytometer at 37 °C. The measurement was based on the change in back-scattered light on abrupt cessation of the erythrocyte suspension (Hardeman et al. 2001). After mixing 420 ll of blood with 110 ll of PVP buffer, a 500-ll mixture was used to measure the aggregation parameters, including aggregation index (AI), aggregation half time (t1/2), and aggregation amplitude (AMP). Statistical analysis Data are expressed as the mean ± standard deviation (SD). Statistical comparisons between groups were evaluated using ANOVA followed by the Student– Newman–Keuls test when the normality and homogeneity of variance assumptions were satisfied; otherwise, ANOVA followed by Student–Newman– Keuls multiple range text were applied. P \ 0.05 was considered significant.
Results Whole blood viscosity The whole blood viscosity measurements are presented in Fig. 1. There were no significantly differences between the two groups at the various shear rates.
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Erythrocyte aggregation The AI, AMP, and t1/2 values are shown in Fig. 3. Although transgenic HO-1G143H mutant mice displayed a trend of increased AI and AMP and decreased t1/2, there was no statistically significant difference between the two groups for AI, AMP, and t 1/2.
Discussion
Fig. 1 The whole blood viscosity in HO-1G143H mutant mice and WT mice. Data are plotted as the mean ± SD (n = 5)
Erythrocyte deformability The erythrocyte deformability measurements are presented in Fig. 2. The elongation index (EI) or integrated EI reflects erythrocyte deformability. EI was significantly reduced in the HO-1G143H mutant mice compared to the WT mice at the shear rates of 600, 800, and 1,000 s-1 (P \ 0.05). In addition, the integrated EI was decreased in the HO-1G143H mutant mice compared to WT mice (P \ 0.05).
The present study demonstrated that a reduction in HO-1 activity in HO-1G143H mutant mice results in impaired erythrocyte deformability. There is no significant difference with respect to erythrocyte aggregation and whole blood viscosity between the HO1G143H mutant mice and the WT mice. Erythrocyte deformability is of fundamental importance for maintaining normal circulation. Reduced erythrocyte deformability contributes to impaired microcirculatory perfusion (Simchon et al. 1987), which results in hypoxia and endothelial dysfunction (Yang et al. 2007; Chen et al. 2012a). Furthermore, a slight decrease in erythrocyte deformability may reduce the rate of entry of erythrocytes into the capillaries and may subsequently impair cellular oxygen delivery and tissue oxygenation (Cicco and Pirrelli 1999). Erythrocyte deformability is regulated by multiple factors, such as oxidative stress and nitric oxide (NO).
Fig. 2 Impaired erythrocyte deformability in HO-1G143H mutant mice. a Erythrocyte deformability (EI) at several shear rates in mice. b Integrated EI in mice. Data are plotted as the mean ± SD (n = 5). #P \ 0.05 versus the WT mice
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Fig. 3 Comparison of erythrocyte aggregation in HO-1G143H mutant mice and WT mice. a AI in mice. b t1/2 in mice. c AMP in mice. Data are plotted as the mean ± SD (n = 5)
Oxidative stress has been reported to decrease RBC deformability in vitro and in vivo (Aydogan et al. 2008; Mohanty et al. 2013). In addition, nitric oxide (NO) is responsible for maintaining normal erythrocyte deformability (Bor-Kucukatay et al. 2003). Decreased NO levels may also contribute to impaired erythrocyte deformability. HO-1 has a potential protective function against oxidative stress (Soares and Bach 2009). HO-1 deficient mice show enhanced levels of oxidative damage (Poss and Tonegawa 1997; True et al. 2007). Furthermore, HO-1 and the metabolic products of heme can also increase the half-life of NO by reducing oxidative stress (Wu and Wang 2005). A previous study indicated that inhibition of HO-1 activity by SnPP results in decreased levels of NOx, which is a stable end product of NO (Ishikawa
123
et al. 2001). Consequently, the impaired erythrocyte deformability in transgenic HO-1G143H mutant mice may result from the enhanced oxidative stress and the consequential reduction in generation and half-life of NO. On the other hand, the dysfunction of abnormal erythrocyte clearance system may also contribute to the impaired erythrocyte deformability in transgenic HO-1G143H mutant mice. It is well known that the spleen serves as the largest filter of blood by initiating immune responses and removing abnormal blood cells (Mebius and Kraal 2005). The structural and mechanical quality of the erythrocytes is ascertained by the mechanical constraint imposed by the meshwork in the red pulp, where old and abnormal erythrocytes that are less deformable are retained and eventually
Transgenic Res
removed by phagocytosis (Huang et al. 2014). However, splenic macrophages that phagocytose senescent erythrocytes die in HO-1-/- mice. The contents released from dying macrophages include nonmetabolized heme, which damages surrounding cells and causes fibrosis in red pulp areas of the spleen, thereby eliminating the normal setting in which recycling of erythrocytes usually occurs (Kovtunovych et al. 2010). Our previous study showed that the HO1G143H mice presented with anemia, enlarged spleen and tissue iron overload, which was similar to HO-1-/- mice (Zhou et al. 2011). So, the abnormal erythrocytes that are less deformable may no longer efficiently removed from the circulation in HO1G143H mice, which contributed to the impaired erythrocyte deformability that we observed. In the present study, there was no significant difference in erythrocyte aggregation, although the transgenic HO-1G143H mutant mice displayed a trend of increased erythrocyte aggregation compared with the WT mice. As mentioned above, HO-1 activity deficient mice showed enhanced levels of oxidative damage. The oxidative damage may accelerate erythrocyte aggregation (Baskurt et al. 1998). One possible explanation for the lack of a significant difference between the two models is that other factors such as erythrocyte properties simultaneously affect erythrocyte aggregation. For example, it has been shown that erythrocyte aggregation can be suppressed by decreased cell deformability (Cicha et al. 1999; Muralidharan et al. 1994; Jovtchev et al. 2000; Reinhart and Singh 1990). In addition, blood viscosity was not significantly different demonstrating that the decreased erythrocyte deformability in transgenic HO1G143H mutant mice had no significant effect on blood viscosity.
Conclusions The current experimental data indicate that a reduction in erythrocyte deformability in transgenic HO1G143H mutant mice. The possible mechanisms may associate with enhanced oxidative stress, decreased NO levels, and the dysfunction of abnormal erythrocyte clearance system in transgenic HO1G143H mutant mice. Our study has limitation in that the mechanism mentioned above were not verified by experimental data. Although the mechanism
underlying this effect remains unclear, our study brings to light the participation of HO-1 in the variations of hemorheology and the limitations will be addressed in continued studies. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 31271001) and the National High Technology Research and Development Program of China (No. 2012AA021902). Conflict of interest
None declared.
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