St. Paul's Hospital, The iCAPTURE Center, University of British Columbia, Vancouver, British Columbia V6Z 1Y6, Canada
Submitted 10 February 2003 ; accepted in final form 19 August 2003
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ABSTRACT |
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excitation-contraction coupling
Earlier studies examining E-C coupling in ASM indicate that contractile activation of airway smooth muscle cells (ASMC) typically involves an increase in intracellular calcium concentration ([Ca2+]i) and the subsequent activation of myosin light chain kinase. Because of this, considerable research efforts have been devoted to the understanding of the mechanism underlying agonist-induced Ca2+ signaling in ASMC. Unlike many of the second messengers involved in intracellular signaling, the Ca2+ signal is unique in the sense that the message is not solely conveyed by a rise in its concentration but also by the spatial and temporal patterns of the Ca2+ signal (18, 19). However, until recently, imaging of [Ca2+]i has been mainly limited to single-cell preparations derived from either nonspecific enzymatic digestion of intact tissue or cell culture. Even though the studies performed in these isolated cell preparations have provided us with great insight into Ca2+ signaling of ASMC (17, 25, 26, 28), there are, unfortunately, some drawbacks to these preparations. It is well documented that the process of cell culture significantly alters the cellular phenotype from the original lineage (10, 13, 34). It therefore may be inappropriate to use observations made in cultured cell preparations to reach conclusions regarding the physiological regulation of intact ASM. Even though the use of freshly digested single-cell preparations avoids complications specific to cell culture, it remains plausible that the loss of intercellular communication (5) and the process of nonspecific enzymatic digestion of surface proteins can significantly alter the physiological characteristics of the ASMC. The findings presented in this paper indeed confirm some of these reservations.
Recently, Bergner and Sanderson (2) examined agonist-induced [Ca2+]i changes of intact ASM that reside in the bronchiolar wall of mice lung slices. They observed that appropriate concentrations of ACh stimulate asynchronous recurring Ca2+ waves in the intact ASMC and cause concomitant narrowing of the bronchiolar lumen. Even though these authors attempted to relate the frequency of the [Ca2+]i oscillations with force generation, the data were inconclusive due to considerable variation and the paucity of data points (2). Thus the relationship between asynchronous wave-like [Ca2+]i oscillations occurring in individual intact ASMC and the overall ASM tissue contraction remains poorly defined.
For the purpose of this study, we have developed a novel technique employing confocal imaging of intact ASM bundles from the porcine trachea attached to a tension transducer. This technique allowed us to measure [Ca2+]i changes from a large number of intact ASMC and simultaneously measure overall force generation by the smooth muscle bundle. The aim of our study was to characterize the ACh-induced Ca2+ signal in intact ASMC within tracheal muscle bundles and relate it to their force generation.
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METHODS |
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Histology and electron microscopy study. Tissues were rinsed in 0.1 M PBS. The tissues were then embedded in Tissue-Tek optimum cutting temperature compound (an embedding medium for frozen tissue specimens), frozen quickly in liquid nitrogen, and stored at -70°C. Eight-micrometer-thick sections were cut at -20°C, collected on slides, and fixed with liquid nitrogen-cooled acetone. The sections were then stained with hematoxylin and eosin. Details of the electron microscopy study have been presented previously, and the procedures, reagents, and chemicals used were identical to what has been previously described (12). Images of the cross sections of the muscle cells were obtained with a Phillips 300 electron microscope.
Simultaneous isometric force measurement and confocal microscopy of Ca2+-induced fluorescence. The clipped muscle strips were loaded with fluo 4-AM (5 µM with 5 µM Pluronic F-127) for 90 min at 25°C and then left to equilibrate for 10 min in normal PSS. They were then immediately mounted onto the custom-made stiff force transducer setup for simultaneous isometric force and [Ca2+]i measurements. The employment of a stiff force transducer, the application of firm clipping to secure the tissue, and the use of small-sized muscle strips are all important for minimizing tissue movements during confocal Ca2+ imaging. Inside the organ bath, one end of the tissue was placed over a stiff metal bar mounted on a micromanipulator for adjustment of muscle strip length, and the other end was connected to the lever arm of a servo-controlled force transducer. The lengths of the mounted muscle strips were set approximately to the intact length. Details of the force measurements employed in this study are similar to what has been described previously (12, 23). Briefly, the servo-controlled force lever system had a force resolution of 10 µN. The analog signals were converted to digital signals via a National Instrument analog-to-digital converter and were recorded by a computer. The details of confocal Ca2+ imaging are also similar to what has been described by us previously (18, 20, 27). Briefly, once the muscle strips were isometrically mounted, the changes in [Ca2+]i were measured using a Noran Oz laser scanning confocal microscope through either an air x60 (numerical aperture 0.7) or an air x20 (numerical aperture 0.45) lens on an inverted Nikon microscope. The tissue was illuminated using the 488-nm line of an argon-krypton laser, and a high-gain photomultiplier tube collected the emission after it passed through a 525/52 band-pass filter. The measured fluo 4 fluorescence level (F525) indicates relative [Ca2+]i, and thus changes in [Ca2+]i are directly reflected by proportional changes in F525. All parameters (laser intensity, gain, etc.) were left unchanged during the experiment. Generally, the acquisition speed was set at 66 ms/frame with two-frame integration resulting in an effective frame rate of 133 ms/frame. Comparisons between recordings made at 66 ms/frame and 133 ms/frame were made when necessary to exclude possible sampling artifacts in the case that the sampling speed was insufficient. Initially, all mounted muscle strips were equilibrated in PSS at 37°C and stimulated twice with high-K+ PSS for 5 min each time. Once the muscle strips were fully relaxed after the second high-K+ stimulation, the experimental protocols were applied.
Data analysis. All confocal image analyses were performed in ImageProPlus using customized routines written in Visual Basic. The muscle strips with recordings that showed significant horizontal and/or vertical movement artifacts were excluded from the study. To obtain data on recruitment of cells during ACh stimulation, a fixed field of view under the x20 lens was chosen, and the number of cells responding with Ca2+ wave(s) was recorded at each concentration of ACh. Recruitment of cells was calculated as a fraction of the number of cells that responded to the highest ACh concentration (100 µM). Further analysis of wave parameters was performed using a three-pixel-wide line along the longitudinal axis of a single cell. The resulting x-t plot revealed the point of origin as well as the progression of the apparent "Ca2+ wave". The frequency of the [Ca2+]i oscillations was determined by counting the number of waves occurring during a period of 40 s. The amplitude of the [Ca2+]i oscillations reflected the difference between the peak F525 of individual Ca2+ spikes in the oscillations and the prestimulation baseline F525. The representative fluorescence traces shown in this report reflect the averaged fluorescence signals from a 3x3-pixel region (1.36 µm2) of the ribbon-shaped ASMC. The fluorescence level (F525) derived in each region is linearly proportional to the [Ca2+]i in that region in such a fashion that any change in [Ca2+]i would be proportionally reflected in the change in F525 level. Such a linear relationship between F525 and [Ca2+]i is not necessarily an absolute since it can be skewed if parameters such as intracellular temperature or pH level change significantly during the course of the experiment. Attempts were made in the experiment to minimize the change of these intracellular temperatures and pH with the use of a precision bath temperature control device and extracellular pH buffer, respectively.
All summarized data are presented as means ± SE. For numerical analysis, all data were analyzed in Excel or Sigma Plot using the appropriate statistical tests. A paired Student's t-test was used for comparisons. A value of P < 0.05 was considered significant. The n values indicated for force development experiments represent the number of tracheal muscle strips (tissues) used, and the n values indicated for the Ca2+ studies represent the number of ASMC analyzed from the specified numbers of tracheal muscle strips.
Solutions and chemicals. Normal PSS containing (in mM) 140 NaCl, 5 KCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES, pH 7.4, at 37°C was used for all studies. High-K+ (80 mM extracellular K+) PSS was identical in composition to normal PSS with the exception of (in mM) 65 NaCl and 80 KCl. Fluo 4-AM and Pluronic F-127, purchased from Molecular Probes, were dissolved in DMSO. Stocks of ACh (Sigma) were prepared in normal PSS, and stocks of nifedipine (Sigma) were prepared in ethanol.
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RESULTS |
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ACh-induced [Ca2+]i signals in intact ASMC of porcine trachea. As revealed by the basal fluorescence level (F525) under confocal microscopy, the intact ASMC of the porcine trachea displays the expected long, ribbon-shaped appearance with a width of 3 µm (Fig. 2A). When stimulated with 5 µM ACh, all the visualized intact ASMC initially responded synchronously with a large Ca2+ wave (Fig. 2A). This initial large Ca2+ wave resulted in an [Ca2+]i elevation that was transient and dissipated within
10-15 s (Fig. 3). After this initial large Ca2+ wave, repetitive intracellular Ca2+ waves were observed traveling along the longitudinal axis of the ribbon-shaped cell. In contrast to the first Ca2+ wave, the subsequent repetitive Ca2+ waves, as depicted in Fig. 3, produced [Ca2+]i elevations that were smaller in amplitude and shorter in duration. As shown in the x-t plot in Fig. 2B, which displays the change in F525 (
[Ca2+]i) over a longitudinal section of the cell over time, a rapid rise in [Ca2+]i was first seen on the left side of this cellular segment and subsequently propagated to the right side of the cellular segment in an apparent wave-like fashion. These apparent Ca2+ waves continue to recur in the same cell for as long as the agonist is present. Figure 2A shows that with the exception of the initial Ca2+ wave, the subsequent oscillatory Ca2+ signals did not occur in a synchronized fashion between neighboring cells. Thus ACh (5 µM) induces asynchronous recurring Ca2+ waves (asynchronous wave-like [Ca2+]i oscillations) in intact ASMC of porcine trachea.
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Relationship between wave-like [Ca2+]i oscillations and force generation. Simultaneous measurements of force and [Ca2+]i showed that 5 µM ACh nearly simultaneously initiated the development of force (tonic contraction) and the generation of wave-like [Ca2+]i oscillations in individual intact ASMC residing within the same muscle strip (Fig. 3). In other words, the induction of the cellular wave-like [Ca2+]i oscillations is temporally closely associated with the induction of tissue force generation, which typically lags 0.5-1.0 s behind the initial generation of the Ca2+ signal (n = 6 tissues). It is important to note that synchronized oscillations in [Ca2+]i usually result in oscillatory force generation, whereas the asynchronous nature of the [Ca2+]i oscillations observed in the intact ASMC accounts for the tonic nature of the force generation by the entire muscle strip.
Furthermore, it was found that increasing concentrations of ACh ranging from 0.01 µM to 100 µM elicited tonic contraction of increasing amplitude by the tracheal smooth muscle (Figs. 4 and 5A). To determine the relationship between the Ca2+ waves and force development, concentration dependences of selected aspects of the wave-like [Ca2+]i oscillations were compared with the concentration dependence of force generation in ACh-stimulated tissues. As shown in the concentration-response curves in Fig. 5B, increasing concentrations of ACh over the concentration range of 0.01 µM to 100 µM are correlated with increasing frequency of [Ca2+]i oscillations, reaching 0.28 ± 0.01 Hz at 100 µM ACh (n = 110 cells from 6 tissues). This observation suggests that the message involved in regulating force development is encoded in the frequency domain of the wave-like [Ca2+]i oscillations. Increasing the concentration of ACh over the lower concentration range from 0.01 µM to 0.1 µM resulted in increased recruitment of cells that display Ca2+ waves (Fig. 5C). It thus appears that incremental recruitment of cells is involved in the modulation of force generation at low-stimulation intensity. A third parameter that was assessed is baseline [Ca2+]i elevation. As shown in Fig. 3, with higher concentrations of ACh and consequently higher frequency of [Ca2+]i oscillations, the interspike trough [Ca2+]i frequently does not return to the prestimulation baseline level. The baseline [Ca2+]i elevation is defined as the average difference between the trough F525 and the prestimulation resting F525. As shown in Fig. 5D, baseline [Ca2+]i elevations were significant and showed concentration-dependent increases only over the higher concentration range of ACh (10-100 µM). It is important to note that the baseline elevation of [Ca2+]i at its highest level was only a quarter of the averaged amplitude of [Ca2+]i oscillations. In contrast to the other assessed parameters, the amplitude of ACh-mediated wave-like [Ca2+]i oscillations did not display a statistically significant concentration dependency (Fig. 5E), indicating that amplitude is not involved in the modulation of the tonic contraction. Finally, the oscillatory Ca2+ signal ([Ca2+]i oscillations) was averaged over time (the entire recording interval) to give the average [Ca2+]i elevation at different ACh concentrations. Figure 5F shows that increasing ACh concentration results also in increasing average [Ca2+]i elevation over time in a concentration-dependent manner.
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Role of L-type voltage-gated Ca2+ channel in ACh-induced wave-like [Ca2+]i oscillations. Ca2+ influx through the L-type voltage-gated Ca2+ channel (VGCC) is known to play an important role in Ca2+ signaling in ASM (4, 6). More specifically, it was reported that ongoing ACh-mediated [Ca2+]i oscillations observed in enzymatically dissociated ASMC from the porcine trachea could be abolished with a high dose of nifedipine (25). To assess the contribution by VGCC, nifedipine was added to ACh-stimulated tissues exhibiting ongoing cellular [Ca2+]i oscillations and tonic contraction. As shown in Fig. 6A, in contrast to what was reported before in enzymatically isolated cells, nifedipine (10 µM) reduced the frequency of ACh (5 µM)-induced wave-like [Ca2+]i oscillations from 0.24 ± 0.02 Hz to 0.17 ± 0.01 Hz (P = 0.002, n = 54 cells from 4 tissues) but did not abolish the ongoing [Ca2+]i oscillations in intact ASMC (even after 15 min of drug treatment). In terms of the average [Ca2+]i levels, nifedipine reduced ACh (5 µM)-induced average [Ca2+]i elevation from 17.6 ± 2.9 F525 units to 11.2 ± 2.1 F525 units (P = 0.02, n = 54 cells from 4 tissues), which is a 36% decrease in average [Ca2+]i elevation. Simultaneously recorded force data revealed that 10 µM nifedipine caused a 32.8 ± 2.9% (P = 0.01, n = 4 tissues) inhibition of the tonic contraction elicited with 5 µM ACh (Fig. 6B). These findings indicate that Ca2+ influx through the L-type VGCC does contribute to, but is nonetheless not essential for, the generation of ACh-induced wave-like [Ca2+]i oscillations and ACh-induced tonic contraction of intact trachea smooth muscle bundles. The 32% reduction in force was similar to the 29% decrease in frequency.
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DISCUSSION |
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The findings presented in this paper reveal that ACh induces asynchronous recurring Ca2+ waves, or wave-like [Ca2+]i oscillations, in the intact ASMC of the porcine trachea. This pattern of Ca2+ signaling has also been recently described in intact ASMC of the terminal bronchiole in a murine lung slice (2). In murine ASMC, ACh initially induced a large wave-like Ca2+ transient, which was followed immediately by the appearance of asynchronous recurring Ca2+ waves. The appearance of the Ca2+ signal coincided with the development of tonic constriction of the terminal bronchiole airway. The frequency of the wave-like [Ca2+]i oscillations reached 0.33 Hz with 100 µM ACh in the murine bronchiole (2), which is comparable with the frequency of 0.28 Hz attained with 100 µM ACh in ASMC from porcine trachea obtained in the present study. Together, these observations demonstrate several important aspects regarding Ca2+ signaling in ASMC at both the tracheal and bronchiolar levels. First, they confirm the ACh-induced wave-like [Ca2+]i oscillations reported in enzymatically dissociated ASMC, although as discussed later, the ability of the wave-like [Ca2+]i oscillations in the intact ASMC to persist in the presence of high-dose nifedipine is a characteristic not shared by its enzymatically isolated counterpart (25). Interestingly, asynchronous Ca2+ waves are seen in these intact ASMC despite the presence of intact gap junctions (Fig. 1). Second, despite the oscillatory nature of the Ca2+ signal at the cellular level, the stimulated constriction of the airway at the tissue level is tonic. This can be explained by the observation that the wave-like [Ca2+]i oscillations do not propagate from cell to cell and are not synchronized between neighboring cells. This property allows for tonic force generation despite intermittent cellular Ca2+ signals. Third, it is important to note that similar asynchronous wave-like [Ca2+]i oscillations have been described in intact vascular smooth muscle cells (VSMC) from a variety of blood vessels (14, 19, 27). It therefore appears that asynchronous wave-like [Ca2+]i oscillations represent a fundamental mechanism for stimulation of tonic smooth muscle contraction.
The main function of ACh-induced wave-like [Ca2+]i oscillations is to signal contractile activation in the ASMC. Our findings provide strong evidence that this, indeed, is the case. The observation that the two processes, wave-like [Ca2+]i oscillations and tonic contraction, are closely associated temporally, with the Ca2+ signals preceding the force generation by 0.5 s, supports this speculation. To further examine how the ACh-induced wave-like [Ca2+]i oscillations at the cellular level can regulate force generation at the ASM tissue level, the concentration dependences of selected aspects of the wave-like [Ca2+]i oscillations were studied and correlated with the concentration dependence of force generation. It was found that the amplitude of the [Ca2+]i oscillations remains constant regardless of the ACh concentration. This is similar to what was reported in both intact bronchial smooth muscle cells (2) and enzymatically dissociated tracheal smooth muscle cells (26). It is, therefore, unlikely that the amplitude domain of the [Ca2+]i oscillations is involved in regulating concentration-dependent contraction. The observed elevation of interspike baseline [Ca2+]i at the high concentrations of ACh (10 and 100 µM) is similar to what has been previously observed by Sieck and coworkers (28) in isolated ASMC. However, in contrast to the isolated ASMC, the average elevation in baseline [Ca2+]i is small (approximately one-quarter) compared with the amplitude of the Ca2+ spikes that constitute the [Ca2+]i oscillations in intact ASMC. Therefore, it is likely that even over the high concentration range of ACh, the degree of contractile activation is more dependent on the frequency Ca2+ waves than the much smaller interspike [Ca2+]i elevations. Furthermore, as shown in Fig. 5, significant interspike (trough) [Ca2+]i elevation occurs only above the 1 µM level of ACh. However, without the input of interspike [Ca2+]i level, 74% of maximal force modulation has already been completed at 1 µM ACh stimulation, apparently achieved through 100% of cell recruitment and 70% of maximal frequency modulation. We thus speculate that the baseline [Ca2+]i elevation observed only at very high ACh concentrations plays little or no role in the physiological regulation of ASM contractility. As mentioned, the recruitment of ASMC initiating Ca2+ waves was observed over the lower concentration range of ACh. This implies that the intact ASMC may have differential sensitivity of the muscarinic receptors to ACh and/or differential effectiveness in the process of initiating Ca2+ waves. Interestingly, a similar observation of differential cell recruitment has been made in VSMC of intact rabbit inferior vena cava (27). In the ASM, it appears that differential recruitment of the intact ASMC initiating Ca2+ waves may be important in regulating ASM contractility over the lower concentration range of ACh stimulation. As for the frequency of the wave-like [Ca2+]i oscillations, they exhibited a concentration dependence that was parallel with force generation. Together, our findings provide strong evidence that the wave-like [Ca2+]i oscillations underlie ACh-mediated tonic contraction of the tracheal smooth muscle. More specifically, with increasing intensity of ACh stimulation, the graded ASM contraction of the intact ASM tissue is achieved first by differential recruitment of the intact ASMC to initiate Ca2+ signals and second by enhancement of the frequency of the wave-like [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. This, however, does not mean that force development in these ASMC is a frequency-sensitive process, although it remains a possibility, since enzymes that are sensitive to the frequency of [Ca2+]i oscillations have been described (7). Alternatively, as shown in Fig. 5F, it is plausible that the graded force generation is achieved on the basis of the average amount of Ca2+ exposed to a cell. In this instance, the average [Ca2+]i over time is determined by the frequency of [Ca2+]i oscillations and the interspike [Ca2+]i elevation. Therefore, by increasing the frequency of the wave-like [Ca2+]i oscillations and elevating the interspike (trough) Ca2+ level, we expose individual ASMC to higher average [Ca2+]i over time.
The spatiotemporal pattern of the Ca2+ signal also provides mechanistic insight into the Ca2+ signal at ASMC (3, 20). In a variety of excitable cells, intracellular Ca2+ waves are initiated by an increase in local [Ca2+]i as a result of Ca2+ influx and/or localized endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+ release that triggers regenerative Ca2+ release from ER/SR (19). The wave-like pattern is a reflection of the SR-mediated Ca2+ release in one cellular region triggering Ca2+-induced Ca2+ release in an adjacent cellular region. Thus the wave-like [Ca2+]i oscillations observed in the intact ASMC of porcine trachea appear to be the result of repetitive "waves" of SR-mediated Ca2+ release as described in VSMC. This is consistent with the findings by Bergner and Sanderson (2) that the wave-like [Ca2+]i oscillations in the bronchiole can be abolished by either predepletion of the SR Ca2+ store or blockade of sarco(endo)plasmic reticulum Ca2+-ATPase (2). However, with repetitive SR Ca2+ release, loss of Ca2+ to the extracellular space is inevitable, and Ca2+ influx must occur to replenish the SR Ca2+ store to maintain ongoing wave-like [Ca2+]i oscillations (20, 25). In enzymatically dissociated ASMC from porcine trachea, it was found that ongoing ACh-mediated [Ca2+]i oscillations cannot persist when L-type VGCC are blocked by 100 nM nifedipine (25). This would suggest that Ca2+ influx through the L-type VGCC is crucial in replenishing the loss of intracellular Ca2+ and in maintaining the SR Ca2+ store. However, in our intact ASMC of the intact porcine tracheal muscle bundle, it was found that a high concentration (10 µM) of nifedipine, maximally effective in blocking VGCC, only partially inhibited the frequency of the ongoing [Ca2+]i oscillations and reduced the tonic contraction by 32.8%. This relatively small inhibitory effect is comparable with what was described previously (15). Because removal of external Ca2+ abolished maintained ACh-induced contraction (22), Ca2+ entry through other pathway(s) is important for replenishing the SR Ca2+ level in ASM. This nifedipine-resistant Ca2+ entry pathway(s) is capable of supporting ongoing wave-like [Ca2+]i oscillations and 67% of the tonic contraction elicited by ACh. Possible nifedipine-resistant Ca2+ entry pathways include receptor-operated channels (ROC), store-operated channels (SOC), reverse-mode Na+/Ca2+ exchange, and other subtypes of VGCC such as the T-type VGCC (1, 9, 16, 20, 30-32). The observed difference between isolated myocytes and intact ASM could be due to enzymatic alteration of both VGCC and ROC/SOC or loss of cell contact.
The findings presented in this paper clearly implicate asynchronous wave-like [Ca2+]i oscillations in the regulation of contractile activity of tracheal smooth muscle. In relation to pulmonary disease, such as asthma, aberrations in Ca2+ signaling of ASM have been somewhat overlooked in the past because of the failure of VGCC blockers to attenuate airway hyperresponsiveness clinically (8, 11). However, the unveiling of asynchronous Ca2+ waves as a fundamental mode of Ca2+ signaling in ASM and our finding that these [Ca2+]i oscillations can persist under maximal blockade of VGCC have shed new light on this matter. Further studies on the mechanisms of wave-like [Ca2+]i oscillations together with an examination on how Ca2+ signaling in diseased airways differ from the typical wave-like [Ca2+]i oscillations observed in healthy airways may reveal novel insights into the pathophysiology of common airway diseases such as asthma.
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DISCLOSURES |
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C.-H. Lee is a recipient of Canadian Institutes of Health Research MD/PhD studentship.
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FOOTNOTES |
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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.
* K.-H. Kuo and J. Dai contributed equally to this work, and C.-H. Lee and C. van Breemen contributed equally to this work.
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REFERENCES |
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