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Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels

Department of Physiology and Cell Biology, College of Medicine and Public Health, Ohio State University, Columbus, Ohio 43210

Submitted 22 May 2003 ; accepted in final form 6 August 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC₅₀ 4.6 nM) with a maximum contraction of 75.1 ± 4.9% at 100 nM compared with that of 60 mM K⁺. Similarly, femoral artery also exhibited a contractile response of 76.0 ± 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 ± 6.6%) and only minimal (3.5 ± 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 µM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar¹]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.

angiotensin II receptors; contraction; endothelial nitric oxide

Over the past several years genetically altered mouse models, including those lacking or overexpressing at least one subtype or one isoform of ANG II receptors, have been generated (3, 4, 9, 13, 14, 17, 18, 24). These genetically altered mice have been used to study the roles of ANG II and other vasoactive substances in cardiovascular diseases such as hypertension (9, 20). However, the physiological role of ANG II in major mouse blood vessels still remains to be explored in detail. It has been shown that in mouse the intrarenal arterioles have a constrictive response to ANG II, suggesting a role for AT1 in regulating the microcirculation (10, 20, 21). On the other hand, the thoracic aorta, a arterial specimen commonly chosen for vascular functional studies in mice, shows only a subtle constrictive response to ANG II (22). Interestingly, AT1 has been shown to exist in the smooth muscle cells of thoracic aorta (22, 24, 31). Therefore, it is imperative to clarify whether the ANG II response is mediated through AT1 in major mouse blood vessels. In addition, AT2, another subtype of ANG II receptors, has also been suggested to exist in blood vessels (1, 12, 28). AT2 has been proposed to mediate vasodilation through endothelial NO release (1, 12, 24, 28). Moreover, ANG II has been found to also mediate endothelial NO release through an undefined mechanism in mouse renal arterioles (20). Thus it would be of interest to determine how AT2 and endothelial NO affect ANG II contractile responses.

In this study ANG II responses in isolated C57/BL6J mouse blood vessels, including abdominal aorta, femoral artery, carotid artery, and thoracic aorta, were studied with isometric force measurements. Experiments were further performed to determine how AT1, AT2, and/or NO contribute(s) to the ANG II responses in mouse blood vessels.

	MATERIALS AND METHODS

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Solutions and chemicals. The ionic composition of physiological saline solution (PSS) was as follows (in mM): 123 NaCl, 4.7 KCl, 15.5 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgCl₂, 1.25 CaCl₂, and 11.5 D-glucose. The 60 mM K⁺-containing PSS was prepared by equimolar replacement of NaCl with KCl. Chemicals such as the NO synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME), ACh, phenylephrine (PE), ANG II, [Sar¹]-ANG II, and PD-123319 were purchased from Sigma (St. Louis, MO). Losartan was purchased from Merck (Whitehouse Station, NJ). All other chemicals were of the highest grade commercially available.

Animals and tissue preparation. Male C57/BL6J mice (8–12 wk) purchased from Jackson Laboratory were euthanized by 95% CO₂ inhalation, in accordance with the animal use protocol of the Animal Research Ethic Committee of Ohio State University. Mouse thoracic aorta, abdominal aorta, carotid artery, and femoral artery were excised rapidly and placed in PSS. Fat and adventitia were mechanically removed under a binocular microscope. The arterial segments were then cut transversely into 1.0-mm-wide vascular rings. These procedures were performed at room temperature.

Isometric force measurement. The method of isometric force measurement for the intact mouse arterial ring was modified from that used for skinned fiber of small vessels as described previously (29, 30). Briefly, the arterial ring was mounted onto two tungsten wires in a 37°C water-circulating tissue bath filled with PSS by passing the tungsten wires through the lumen of the arterial ring. One of the wires was fixed, and the other was connected to a force transducer (AE 801, Horten). During the equilibration period, the tissues were stimulated with 60 mM K⁺ every 15 min (5 times) and the resting tension was increased in a stepwise manner. After equilibration, the resting tension was adjusted to 300 mg, at which the maximal 60 mM K⁺ response was obtained. Under such condition, 60 mM K⁺ typically generates 410, 370, 280, or 170 mg of force in tissue preparations from mouse thoracic, abdominal aorta, femoral artery, or carotid artery, respectively.

Experimental protocols. The physiological studies were conducted in blood vessels with intact endothelium at 37°C. ANG II was used only once in each specimen and was administered 15 min after the final 60 mM K⁺ contraction had been relaxed with PSS. The force development caused by ANG II was expressed as a percentage of that obtained with 60 mM K⁺, assuming the values in PSS (5.9 mM K⁺) and 60 mM K⁺ to be 0% and 100%, respectively. The temporal parameter of t_p (time to reach the peak) or t_d (the additional time for the force to descend to one-half of the peak) was measured to evaluate the time course of ANG II-induced responses. Unless otherwise indicated, L-NAME, losartan, or PD-123319 was applied 5 min before ANG II was added. In controls, these agents were replaced with their solvents. The intactness of endothelium was tested by ACh (10 µM)-induced relaxation in contraction caused by 10 µM PE at the end of each experiment.

Detection of AT1 mRNA levels. Aortas (abdominal or thoracic) were used for total RNA extraction. Tissues were prepared as described in Animals and tissue preparation with the exception that they were not cut into arterial rings. RNA preparation and RT-PCR were performed with an Absolutely RNA RT-PCR Miniprep Kit according to the manufacturer's manual (Stratagen, La Jolla, CA). Primers for AT1 receptor were designed from common sequences of AT1 isoforms as previously described (31): 5'-CCAAAGTCACCTGCATCATC-3' (PCR sense) and 5'-CACAATCGCCTAATTATCCTA-3' (RT and PCR antisense). RT was performed with 200 ng of total RNA in a volume of 20 µl. The protocols for PCR were as follows: 94°C for 30 s, 56°C for 60 s, and 72°C for 60 s (30 cycles). The expected size of PCR products was 305 bp. To determine the specificity of RT-PCR reactions, the PCR mixture obtained from mouse abdominal aorta was precipitated with 50 µl of 2-propanol and washed with 100 µl of 70% ethanol. After being air dried, PCR products were dissolved in 20 µl of water and digested with 10 U of BstXI (New England Biolabs, Beverly, MA) at 55°C for 3 h. The digestion yields fragments of 149 and 156 bp for AT1 PCR product based on the mouse cDNA sequences. Detection and quantification of PCR products were performed with a Biochemi System (UVP, Upland, CA). In addition, mRNA for -actin was also amplified by RT-PCR to serve as an internal control (expected product size 509 bp), which was performed as previously described (16).

Statistics. The EC₅₀ value, a concentration at which 50% of maximum response was obtained, was determined from the concentration-response curves fitted to a four-parameter logistic model (7). Data are expressed as means ± SE. Student's t-test or an analysis of variance with multiple comparisons was used to determine the statistical significance. P < 0.05 was considered to indicate statistical significance.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
ANG II-induced vasoconstriction in mouse blood vessels. The role of ANG II in mouse vascular physiology has not been explored in detail. To determine whether ANG II mediates similar responses in different mouse vessels, we studied ANG II-induced contractions in major blood vessels of C57/BL6J mice. First we examined the abdominal aorta. As shown in Fig. 1A, in response to 1, 10, or 100 nM or 1 µM of ANG II, the mouse abdominal aorta developed a contraction of 13.5 ± 4.2%, 49.6 ± 5.1%, 75.1 ± 4.9%, or 70.8 ± 3.1%, respectively, compared with that of 60 mM K⁺, with an EC₅₀ of 4.6 nM. Figure 1B demonstrates a representative recording showing the maximum contractile response in abdominal aorta induced by 100 nM ANG II, which was biphasic in property (see Table 1 for t_p and t_d values), as seen in other species (25).

View larger version (13K): [in this window] [in a new window]   Fig. 1. ANG II-induced contraction in isolated mouse abdominal aorta. A: summary of concentration-responses induced by ANG II in mouse abdominal aorta with intact endothelium. The force development is expressed as a percentage of that with 60 mM K+. Values are expressed as means ± SE (n = 5). B: representative recording showing the maximal contraction induced by 100 nM ANG II in mouse abdominal aorta with intact endothelium.

The maximum responses of femoral artery, carotid artery, and thoracic aorta to ANG II (100 nM) were examined next. As shown in Fig. 2, ANG II (100 nM) also induced a biphasic contraction in the femoral and carotid artery (see Table 1 for t_p and t_d values). The contraction in the femoral artery was not significantly different from that in abdominal aorta (76.0 ± 3.4%; Fig. 2, A and D). On the other hand, in carotid artery the ANG II response was much smaller (24.5 ± 6.6%) compared with the abdominal aorta or femoral artery (Fig. 2, B and D). Furthermore, the maximum contractile response to ANG II (100 nM) was only barely detectable (3.5 ± 0.31%, Fig. 2, C and D) in the thoracic aorta.

View larger version (11K): [in this window] [in a new window]   Fig. 2. ANG II-induced contractions in isolated femoral, carotid, and thoracic vessels with intact endothelium. A–C: representative recordings showing the contraction induced by 100 nM ANG II in femoral artery (A), carotid artery (B), and thoracic aorta (C). D: summary of 100 nM ANG II-induced responses in abdominal aorta (AA), femoral artery (FA), carotid artery (CA), and thoracic aorta (TA). The force development is expressed as a percentage of that with 60 mM K+. Values are expressed as means ± SE (n = 5). P < 0.05: *compared with abdominal aorta, #compared with femoral artery.

Effect of L-NAME on ANG II-induced responses. Endothelial NO release mediated through AT2 or an undefined mechanism has been proposed to be a part of ANG II response (20, 28). To observe how this component would affect ANG II contractile response, the effect of L-NAME was examined. As shown in Fig. 3A, in the presence of 1 mM L-NAME, the contractions caused by 1, 10, and 100 nM ANG II in abdominal aorta (7.0 ± 3.5%, 55.4 ± 5.7%, and 74.1 ± 1.1%, respectively, compared with that of 60 mM K⁺) were not significantly different from those of control. Similarly, 1 mM L-NAME did not significantly change contraction induced by 100 nM ANG II in femoral artery (75.1 ± 3.6%), carotid artery (24.6 ± 4.5%), or thoracic aorta (5.1 ± 4.5%) (Fig. 3B). To further evaluate whether the concomitant NO release modulates the time course of ANG II response, the values of t_p and t_d of 100 nM ANG II-induced contraction in abdominal aorta in the presence of 1 mM L-NAME were determined. Neither of these two parameters (t_p 127.5 ± 9.7 s, t_d 228.8 ± 24.1 s; n = 4) was significantly different from that in the absence of L-NAME. In contrast, 1 mM L-NAME abolished an average of 80–85% relaxation caused by 10 µM ACh on 10 µM PE-induced contraction in major mouse blood vessels (Fig. 3C, representative recordings from the abdominal aorta). Therefore, the concomitant endothelial NO release does not appear to significantly affect the ANG II contractile responses.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Effect of NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) on ANG II-induced contractions. A: summary of the effects of L-NAME on ANG II response in abdominal aorta with intact endothelium. , Responses from control (n = 5); , responses from tissues pretreated with L-NAME (n = 3–4). B: summary of the effects of L-NAME on ANG II response (100 nM) in femoral artery (FA), carotid artery (CA), and thoracic aorta (TA). Open bars, control tissues (n = 5); hatched bars, tissues pretreated with L-NAME (n = 3–4). Values are expressed as means ± SE. C: representative recordings showing the effect of L-NAME on ACh-induced relaxation on phenylephrine (PE)-induced contraction in abdominal aorta with intact endothelium. Top: a representative endothelium-dependent relaxation induced by 10 µM ACh in mouse abdominal aorta. Bottom: relaxation in the presence of 1 mM L-NAME. Force is expressed as a percentage of that with 60 mM K+.

Effects of specific AT1 or AT2 antagonists on mouse vessels. To further evaluate the role of ANG II receptors in mouse blood vessels the effect of PD-123319, an AT2 antagonist, and losartan, an AT1 antagonist, were examined. As shown in Fig. 4A, in the presence of 100 nM PD-123319, 100 nM ANG II-induced contraction was not significantly affected either in abdominal aorta (71.7 ± 3.0% vs. 73.3 ± 3.8%) or carotid artery (22.9 ± 3.7% vs. 22.3 ± 3.8%). The effect of PD-123319 was not enhanced by increasing the concentration or prolonging the incubation (data not shown). In contrast, in the presence of losartan (100 nM), 100 nM ANG II-induced contraction was significantly decreased to 22.3 ± 3.8% (Fig. 4A) in abdominal aorta and almost completely inhibited in carotid artery (1.3 ± 0.4%; Fig. 4A). Furthermore, the 100 nM ANG II-induced contraction in abdominal aorta was also completely inhibited when 10 µM losartan was used (Fig. 4B).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Effect of ANG II receptor antagonist on major mouse blood vessels with intact endothelium. A: summary of the effects of 100 nM of AT1 antagonist losartan or AT2 antagonist PD-123319 on the responses induced by 100 nM ANG II in abdominal aorta (AA) and carotid artery (CA). Open bars, values from control; hatched bars, values in the presence of 100 nM PD-123319; filled bars, values in the presence of 100 nM losartan. Force development is expressed as a percentage of that with 60 mM K+. Values are expressed as means ± SE (n = 5). *P < 0.05, compared with control. B: representative recording (from 3 identical experiments) showing the complete inhibition of 100 nM ANG II-induced response by 10 µM losartan in abdominal aorta.

Action of AT1-selective agonist [Sar¹]-ANG II on mouse blood vessels. The above results indicated that ANG II responses in major mouse blood vessels are solely mediated by the AT1 receptor. Therefore, it is expected that an AT1-selective agonist should have a similar effect on mouse vessels. To test this hypothesis, the effect of an AT1 agonist, [Sar¹]-ANG II, on mouse vessels was examined. As shown in Fig. 5, 100 nM [Sar¹]-ANG II induced a contraction of 71.3 ± 2.8% (n = 3) or 19.8 ± 3.7% (n = 3) in abdominal aorta or carotid artery, respectively, which was not significantly different from that of 100 nM ANG II.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effect of AT1 agonist [Sar1]-ANG II on mouse blood vessels with intact endothelium. Representative recordings demonstrating the contraction in the presence of 100 nM [Sar1]-ANG II in abdominal aorta (A) and carotid artery (B) are shown.

AT1 mRNA expression in mouse thoracic and abdominal aorta. To explore the molecular basis for the different ANG II responses in mouse vessels, AT1 mRNA expression levels in thoracic and abdominal aorta were examined with RT-PCR. As shown in Fig. 6A, PCR products of the expected size (305 bp) could be clearly seen in both thoracic and abdominal aorta. On the other hand, densities (normalized to that of -actin) of PCR products in thoracic aorta and abdominal aorta were in a ratio of 2.5 ± 0.6 to 10 (n = 4; P < 0.05). To confirm the specificity of RT-PCR reaction, the PCR products obtained from abdominal aorta were further digested with BstXI, which cuts AT1 PCR products at the site of nucleotide 149. As shown in Fig. 6B, after digestion with BstXI, the PCR products appeared as a band of 150 bp, consistent with the sizes (149 and 156 bp) expected.

View larger version (60K): [in this window] [in a new window]   Fig. 6. AT1 mRNA levels in mouse thoracic and abdominal aorta. A, top: levels of AT1 mRNA amplified by RT-PCR in mouse thoracic (lane 1) and abdominal (lane 2) aorta. Bottom: internal control of PCR products (509 bp) for -actin mRNA level in mouse thoracic (lane 1) and abdominal (lane 2) aorta. B: digestion of PCR product obtained from mouse abdominal aorta with BstXI, which was expected to produce fragments of 149 and 156 bp. Lane 1, AT1 PCR products digested with BstXI; lane 2, undigested. M, 100 bp-ladder size makers [top to bottom: 500–100 bp for the demonstrations of AT1 and 600 to 400 bp for -actin (New England Biolabs, Beverly, MA)].

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
To date, the ANG II responses in mouse blood vessels has not been well defined. The mouse thoracic aorta, a common specimen for studying vascular function in mouse, has been demonstrated to have little response to ANG II (22), whereas other vessels remain largely unstudied. This may have been because of the size of mouse blood vessels, which makes isometric force measurements difficult to perform. To address this problem, we have modified the apparatus originally used to measure isometric force in skinned fiber preparations of small vessels. With this apparatus, the force developments of various intact mouse blood vessels could be clearly assessed above small baseline oscillations (in most cases, only 1–2% compared with the contractions induced by 60 mM K⁺). Therefore, this approach was used to study ANG II responses in major blood vessels of C57/BL6J mice, a strain that has been used widely to generate transgenic models.

Results from the abdominal aorta and femoral artery indicate that ANG II is a strong constrictor in major mouse blood vessels. However, the contractile response to ANG II is not the same among major mouse blood vessels. Similar to a previously reported result (22), ANG II is poorly effective in thoracic aorta, in which the contractile response to 100 nM ANG II was minimal compared with that of abdominal aorta or femoral artery (Fig. 2). In addition, our data also demonstrate that in carotid artery, a peripheral artery, the contraction induced by 100 nM ANG II was only about one-third of that in abdominal aorta or femoral artery (as percentage of that induced 60 mM K⁺; Fig. 2). However, the maximum PE-induced contractions (in percentage of that induced by 60 mM K⁺; data not shown) in mouse thoracic aorta and carotid artery were comparable to those observed in abdominal aorta (Fig. 3C). In addition, a previous study also found that, in contrast to ANG II, other agonists, such as norepinephrine and PGF₂, induced potent contractile responses in mouse thoracic aorta (22). Therefore, the diminished ANG II contractile response in mouse thoracic aorta or carotid artery is not likely to be caused by physical properties such as wall thickness.

ANG II has also been proposed to cause endothelial NO release through either AT2 or an undefined mechanism (2, 20, 26, 28). Therefore, it seems reasonable that the contractile response, especially in thoracic aorta and carotid artery, might be masked under the influence of AT2/NO. However, pretreatment with 1 mM L-NAME, which abolished the endothelial relaxation caused by 10 µM ACh (Fig. 3C), did not significantly affect ANG II-induced contractions in major mouse blood vessels (Fig. 3). In addition, the AT2-specific antagonist PD-123319 did not enhance the ANG II-induced contractions in abdominal aorta and carotid artery as well (Fig. 4). Therefore, it appears that neither AT2 or endothelial NO affects ANG II contractile response, which is consistent with studies reported previously in wild-type mouse aorta [site not specified (24) or thoracic aorta (22)]. It must be emphasized that the biphasic property of ANG II-induced responses was not due to the influence of AT2/NO but rather to the desensitization of ANG II receptors (AT1), as pointed out previously (19, 23). In agreeing with this proposal, we also found that the NOS inhibitor L-NAME did not significantly affect the t_p or t_d value of 100 nM ANG II-induced contractions in abdominal aorta.

The role of AT1 in ANG II contractile response is clearly demonstrated by a complete inhibition with losartan, an AT1 antagonist (Fig. 4). Furthermore, a lack of effect for AT2/NO suggests that AT1 may be the sole determinant of ANG II response in the vessels studied. This is also supported by the action of [Sar¹]-ANG II, an AT1 agonist, which mimicked ANG II in mouse abdominal aorta and carotid artery (Fig. 6). To explore the molecular bases for the difference in ANG II response among major mouse vessels, levels of AT1 mRNA in mouse thoracic and abdominal aorta were examined with RT-PCR as previously reported (31). As demonstrated by the quantities of PCR products, the mouse thoracic aorta, which exhibits only a minimal response to ANG II, has a significantly lower level of AT1 mRNA than abdominal aorta (Fig. 6). These results allow us to suggest that the difference in ANG II response among major mouse blood vessels is due to the varied AT1 receptor levels.

The findings of this study are in contrast to a study performed on the intrarenal arterioles, in which the disruption of endothelial NOS gene caused a stronger vasoconstriction by ANG II, suggesting a role of ANG II-induced NO in regulating the microcirculation (20). Although the mechanism of this ANG II-elicited NO release in mouse renal microcirculation has not been defined (20), it is noteworthy that a potential endothelial NO mediator, AT2, is distributed in a tissue-dependent manner. Although AT2 was shown to be present in intrarenal vasculature in species such as human and rat (1), it is not present in major mouse blood vessels such as aorta (24). In addition, most AT2-mediated NO-dependent vasodilations were reported in the microcirculation (12, 28). Therefore, it is likely that there is a difference of endothelial NO involvement in ANG II response between the intrarenal arterioles and the major mouse blood vessels. This difference may partially explain why the deletion of AT2 gene in mice induced a stronger pressor response to ANG II (11, 13), whereas our study showed that AT2 or a concomitant NO release did not significantly mediate ANG II response in large mouse blood vessels.

One of the most important findings of this study is that ANG II induces differential contractions among major mouse blood vessels. It has been reported that in mouse the blood concentration of ANG II is 0.1 nM (5). At this level, ANG II alone might not induce vasoconstrictions in vessels studied. However, under certain critical conditions, such as shock or imbalance of blood volume, RAS activities could be significantly enhanced, thus increasing the amount of ANG II released into blood (27). In addition, ANG II could also be locally produced in the vasculature in situ (8). Therefore, it is possible for ANG II to differentially affect contractility among major mouse blood vessels.

In summary, this is the first study to systemically examine ANG II responses among major mouse vessels with isometric force measurements. We have found that ANG II induces differential contractions among major mouse vessels. This is possibly due to the differences in AT1 receptor level, as suggested by the measurements of AT1 mRNA with RT-PCR. In addition, our data also indicate that the ANG II contractile responses in the vessels studied were not affected by AT2 or ANG II-mediated endothelial NO release.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-38355-15 (to M. Periasamy). Y. Zhou is an American Heart Association (Ohio Valley) Postdoctoral Fellow.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Jonathan Davis for critical reading and comments on the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Periasamy, Dept. of Physiology and Cell Biology, Ohio State Univ. Coll. of Med., 304 Hamilton Hall, 1645 Neil Ave, Columbus, OH 43210 (E-mail: periasamy.1{at}osu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

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