Published online before print June 12, 2008, doi: 10.1161/CIRCRESAHA.107.168567
© 2008 American Heart Association, Inc.
Molecular Medicine |
Endothelial Nitric Oxide Synthase Gene Expression During Murine Embryogenesis
Commencement of Expression in the Embryo Occurs With the Establishment of a Unidirectional Circulatory System
From the Renal Division and Department of Medicine (A.-M.T., J.A.S., G.B.R., M.L., A.K., B.M.S., P.A.M.), St. Michael’s Hospital; Department of Medicine (A.-M.T., J.A.S., G.B.R., M.L., A.K., A.C.S., P.A.M.) and Heart & Stroke/Richard Lewar Centre of Excellence in Cardiovascular Research (S.L.A., M.I.C., P.A.M.), University of Toronto; Samuel Lunenfeld Research Institute (Y.-Q.Z., S.L.A.), Mount Sinai Hospital; Mouse Imaging Centre (Y.-Q.Z., S.L.A.), Hospital for Sick Children; Department of Obstetrics and Gynecology (S.L.A.), Samuel Lunenfeld Research Institute, Mount Sinai Hospital; and Toronto General Research Institute (S.-N.Z., M.I.C.), University Health Network, Toronto, Canada.
Correspondence to Philip A. Marsden, MD, Rm 7358, Medical Sciences Building, University of Toronto, 1 Kings College Circle, Toronto, ON, M5S 1A8, Canada. E-mail p.marsden{at}utoronto.ca
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Abstract |
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To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with β-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.
Key Words: angiogenesis • embryogenesis • gene transcription • reporter gene • shear stress
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many important biological functions of endothelial NO synthase (eNOS)-derived NO have been described in adult mammals. Although NO is an essential endogenous messenger in the regulation of vascular smooth muscle tone and organ blood flow, our understanding of the role of NO during development is limited. There is a paucity of information about the regulatory factors that contribute to the establishment and remodeling of blood flow during mammalian embryogenesis. Given the importance of eNOS in regulating vascular tone in the adult, we investigated its expression during development of the embryonic circulatory system.
Prior in vitro studies of the human eNOS promoter have identified functionally important conserved regulatory domains.1–4 In the proximal promoter, nucleoprotein complexes containing the Sp1 and Ets family members Maz and YY1 form on positive regulatory domains I and II and are requisite for constitutive expression of eNOS.1 Sequence inspection of the murine eNOS promoter has revealed substantial conservation of regulatory domains that are important for constitutive expression.2 In vivo investigations have also used insertional eNOS promoter–reporter transgenic lines.5,6 The eNOSpromnlsLacZ line uses 5.2 kb of the 5' flanking region of the murine eNOS promoter to direct transcription of the reporter gene LacZ, encoding β-galactosidase5 that is highly restricted to endothelial cells of medium- and large-size arteries in adult mice. These in vivo models provide a conceptual framework for examining the homeostatic and pathophysiological regulation of eNOS expression.
It is not known when eNOS is expressed during embryonic development. Clearly eNOS-derived NO is functionally important during the later stages of development of the circulatory system, because eNOS-knockout mice exhibit abnormal development of aortic valves,7,8 congenital atrial and ventricular septal defects,9 and abnormal pulmonary vascular development.10 Confounding these studies is the realization that expression and function of the 3 distinct mammalian NOS isoforms are not completely independent and that deletion of 1 isoform may be compensated for by modulation of expression of another.11 Expression of other endothelial cell-specific gene products, such as the tyrosine kinase receptors vascular endothelial growth factor receptor (VEGF-R1), VEGF-R2, and Tie2/Tek, have been well studied during development and are critically important for early blood island formation, vasculogenesis, and angiogenesis.12 In the present work, we comprehensively examined where and when eNOS is expressed during murine development, focusing on the primordial vascular network. We report that eNOS transcription in the murine embryo occurred later than other endothelial cell-specific genes in endothelial cell ontogeny. Thus, eNOS expression follows, rather than precedes, the establishment of unidirectional circulation of blood.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online supplement at http://circres.ahajournals.org.
Generation of Transgenic Mice
A detailed description of the insertional promoter–reporter eNOSpromnlsLacZ transgenic strain has been reported previously.5 We generated a new eNOSGFP promoter–reporter strain to allow in vivo observation of reporter expression. Flk1 heterozygous mutant mice, in which the LacZ reporter is under the transcriptional control of the endogenous flk1 promoter, have been described previously.13
Transgene Expression at Timed Embryonic Stages
We generated litters of hemizygous embryos by mating wild-type female mice to hemizygous males. The postcoital morning, when a vaginal plug was observed, was deemed to be embryonic day (E)0.5. At the specified time points, embryos were excised for fixation and X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) staining.
Expression of green fluorescent protein in whole, live eNOSGFP embryos was visualized and imaged under a Leica MZ 95 dissecting microscope fitted with a MAA-03 light source. Digital 36-bit color images were acquired and processed off-line.
Ultrasound Biomicroscopy–Doppler
Timed pregnant mice (eNOSGFPxC57BL6/SJL) were studied over E8.5 to 10.5. The embryonic primitive heart tube (E8.5 and E9.5) or dorsal aorta (E9.5) were imaged in real-time using an Ultrasound Biomicroscope. The 2D image was used to position the Doppler sample volume at the site of interest and Doppler flow velocity spectra were recorded.14
In Situ cRNA Hybridization
A 992-nt antisense probe detecting wild-type eNOS mRNA and a 1008-nt control sense probe was synthesized using T3 and T7 polymerase, respectively, and were labeled with [35S]-UTP. In situ hybridization was performed as previously described.15
Immunostaining of Embryos
Immunofluorescence staining of eNOS protein in embryos was performed using a tyramide signal amplification system. Images were obtained using a Bio-Rad 1024 confocal microscope.
Data Analysis
Unless otherwise stated, experiments were performed at least 3 times. Comparisons of transfections were made with ANOVA. Probability values of <0.05 were considered statistically significant.
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Results |
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Cardiovascular Expression of eNOS During Development
To evaluate transcriptional activity of the eNOS gene during mammalian development, we used eNOSpromnlsLacZ promoter/reporter transgenic mice. Expression of the LacZ reporter was first evident in mouse embryos at E9.5 (Figure 1A and 1B). To define embryonic stages, we correlated somite number (S) with the onset of β-galactosidase staining. LacZ expression was first observed at an average somite count of 19.8±2.7 S (mean±SEM, 20 embryos), corresponding to E8.5 to E9.5 (11S to 20S) and E9.5 to E10.5 (21S to 30S).16
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Using β-galactosidase activity to indicate eNOS transcription, we noted strong expression at E9.5 in the vascular endothelium of the dorsal aorta (Figure 1C) and intersomitic blood vessels (Figure 1B) and in the endocardium (Figure 1A and 1D). At E11.5, LacZ expression was strong in the descending aorta (Figure 1C) and the endocardium overlying the endocardial cushion of the developing outflow aortic valves (Figure 1D and 1G). Extraembryonic LacZ expression was particularly robust at E13.5 in the endothelial layer of the yolk sac (Figure 1E and 1F). β-Galactosidase activity was generally evident in the endothelial lining of all major embryonic vessels at E11.5. Transgene expression continued to be evident throughout blood vessel remodeling (Figure 1H). The major arteries of maturing solid organs exhibited discrete expression of the transgene at E14.5 in the kidney (Figure 2A) and the brain (Figure 2B). β-Galactosidase staining in the endocardium decreased with increasing embryonic age (Figure 2C).
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To assess transcription of the eNOS gene in vivo, we generated a new eNOS promoter–reporter transgene using enhanced green fluorescent protein (GFP) (Figure 3A).17 The MueNOSGFP transgenic promoter–reporter line allowed reporter expression to be visualized in vivo. In utero ultrasound biomicroscopy (UBM) imaging and Doppler were used to correlate the onset of the embryonic heartbeat and cardiac blood flow with the initiation of reporter expression in the early mouse embryonic circulation. UBM studies indicated pulsatile Doppler blood velocity signals in the primitive heart tube in 82% of embryos at E8.5 (n=11), before detection of LacZ expression in the embryonic circulation. Directional flow was detected in the primitive heart loop and/or dorsal aorta in 97% of E9.5 embryos (n=29), and GFP expression was evident in all of the transgene-positive animals (Figure 3B and 3C). GFP reporter expression was prominent in the aortic arch, brachial arch arteries, intersomitic vessels, and endocardium at E9.5 (Figure 3D). In adult mice, as in development, expression of the GFP reporter was restricted to arterial (foreground vessel) versus venous (background vessel) vascular endothelium (Figure 3E). It is interesting to note that expression of the reporter was different at the flow divider of the artery versus the lateral shoulders of the bifurcating feeder vessel, consistent with the recent findings of Won et al.18 Transgene expression was not detected in neuroectodermal or other mesodermal tissues.
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In contrast to other endothelial cell-restricted genes that are expressed at E7.5,13 the onset of eNOS expression in vascular endothelium is a late event in endothelial ontogeny. To compare the expression pattern of VEGF-R2 and eNOS at E7.5 and E8.5, we used mice that express the LacZ gene from the endogenous Flk1 promoter.13 eNOSpromnlsLacZ transgenic mice exhibited no β-galactosidase staining at E7.5 or E8.5 (Figure 4), in contrast to the strong expression of the LacZ gene in primitive hematopoietic blood islands and the rudimentary aorta of heterozygous Flk1+/LacZ mice at the same stages. Thus, the eNOS gene is not expressed in the primordial vasculature of mammalian embryos at the earliest stages of vasculogenesis and angiogenesis. In murine blood vessels, the expression of the β-galactosidase reporter mRNA can be detected before β-galactosidase activity is evident.19 The strong expression of the LacZ gene at earlier developmental stages in the Flk1+/LacZ mice makes it unlikely that significant reporter gene expression in the eNOS reporter mice was missed at these early stages.
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A notable example of nonendothelial transgene expression was observed in the apical ectodermal ridge (AER). The AER is a developmental structure found at the most distal edge of the developing limb that is mechanistically implicated in limb appendage proximodistal growth.20,21 Discrete β-galactosidase staining was evident at the AER in developing upper and lower limbs (Figure 5). Transgene expression in the AER was first evident at E9.5 in the primordial upper limbs. At E13.5, expression became restricted to the outermost edges of each digit and was completely absent in the cleft web regions between digits (Figure 5A, left inset). The morphology of the positively stained cells in the AER was of a columnar type (Figure 5A, right inset). We also observed strong transgene expression at the macro- and microscopic level in developing skeletal muscle myocytes in the proximal regions of the four limbs at E14.5 to E17.5 (Figure 5B and 5C).
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Because mRNA transcripts derived from promoter/reporter transgenic genomic constructs do not contain important potential eNOS cis-RNA regulatory sequences, and solely reflect transcriptional control pathways, we examined the distribution of native eNOS mRNA transcripts using in situ cRNA hybridization, RNase protection, and real-time RT-PCR throughout mouse embryogenesis. Representative studies from whole embryo in situ [35S]-labeled cRNA hybridization at E9.5, E11.5, and E14.5 are shown in (Figure 6A through 6D). Sense control probes showed no signal (Figure 6E through 6H). The hybridization signals for antisense eNOS cRNA probes, but not sense eNOS cRNA probes, coincided exactly with the expression profile observed for reporter transgenes. At E9.5, the eNOS mRNA transcript was expressed specifically in the endothelium of developing blood vessels, including the arteries of the aortic arch, intersomitic arteries, vessels of the limb bud, and vessels at the periphery of the neural tube and the brain. The endocardium of the developing heart also exhibited positivity at E9.5 (Figure 6A). At E11.5, eNOS mRNA was detected in the aorta, in the now larger blood vessels of the developing pharynx, and in the central vessels of the limb bud. The transcript was also detected in the umbilical vessels and the basilar artery of the hindbrain and demonstrated continued high-level expression in the intersomitic arteries. At E11.5, the endocardium of the atria and ventricles also exhibited positivity (Figure 6B). At E14.5, detection of eNOS transcript was attenuated throughout the embryo (Figure 6C and 6D) and qualitative examination suggested that expression was becoming restricted to medium- and large-sized blood vessels, comparable with adult mice. Areas of positivity included blood vessels of the hindlimb in the developing musculature, vessels of the stomach, intestine, and lung. Endothelial cells of the blood vessels surrounding the forebrain and hindbrain, including the basilar artery, were positive for eNOS signal. eNOS mRNA was still evident in the endocardium of the atria and ventricles at E17.5. In general, the level of mRNA expression decreased from E9.5 to E14.5. Consistent with the profile of reporter transgene expression, in situ cRNA hybridization revealed eNOS mRNA transcripts in the AER at E11.5 (data not shown).
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We performed immunohistochemistry for eNOS in developing embryos to determine whether eNOS protein and mRNA expression were congruent with reporter gene expression. The endothelial cells lining dorsal aorta were immunopositive for eNOS protein at E9.5 (Figure 7A and 7B). These same cell types were also positive for CD31 (platelet endothelial cell adhesion molecule) (data not shown). At E13.5, serial sections of a developing forelimb (Figure 7C and 7D) revealed expression of eNOS protein in the AER. These findings confirm that eNOS mRNA, reporter genes, and protein expression are congruent.
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Discussion |
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We examined the expression profile of eNOS throughout murine embryonic development by quantifying the native eNOS mRNA and protein expression and assessed transcription of the eNOS gene using eNOSpromnlsLacZ and eNOSGFP reporter transgenic mouse strains. Given the importance of eNOS in the regulation of vascular tone in the adult, we focused primarily on the circulatory system of the embryo. Our work did not address eNOS expression at the earliest stages of mammalian development (ie, in gametes, at the time of egg fertilization, during the first few cell divisions of life) or in extraembryonic tissues. We were especially motivated to define the time of onset of eNOS transcription with respect to the initiation of circulatory flow in the embryo. This investigation clearly defines that the transcriptional activity of the eNOS gene follows, rather than precedes, the onset of circulatory flow in the developing mammalian embryo. Thus, the eNOS gene is not expressed in the primordial vasculature of mammalian embryos at the earliest stages of vasculogenesis and angiogenesis. Consequently, eNOS is a late endothelial cell marker. This contrasts with the onset of expression and functional importance of early endothelial genes (ie, Flk-1 (E7.0),22 Flt-1 (E7.5),23 Tie1 (E8.0), Tie2 (E7.5),24 thrombomodulin (E8.5),25 von Willebrand factor (E8.5),26 or VE-cadherin (E7.5).27
The murine endocardial tube develops at E8.0 and is rapidly joined to the dorsal aorta. The onset of rhythmic cardiac contractions and movement of blood can be detected by Doppler velocity waveforms before cardiac looping, at E8.5. At E9.5, the murine heart rate averages 100 to 110 bpm and is associated with both the inflow and outflow of blood. Septation of the heart begins at E11.5 and by E13.5 the heart exhibits a fully septated structure with well-defined chambers. Our observation of transgene expression in primordial endocardial cushions, eg, the future outflow aortic valve region at E11.5, is important in understanding a potential role for eNOS-derived NO in cardiac development, particularly because eNOS–/– mice are predisposed toward developmental defects of the aortic valve.8
The finding that flow preceded transcription of eNOS in vascular endothelial cells of the embryo during mammalian development highlights the functional importance of hemodynamically mediated alterations in endothelial gene expression. Endothelial cells in the cardiovascular system exhibit multiple phenotypes in response to the complex flow patterns that occur in vivo. It is not clear which of the different biomechanical stimuli are relevant to patterns of gene expression in a living mammal, especially during development. Endothelial genes are regulated differentially by unique hemodynamic forces, ie, laminar shear stress, turbulent shear stress, and disturbed flow. eNOS is a well-known example of an endothelial gene whose expression is modified by flow patterns, especially arterial levels of laminar shear stress. Microarray transcriptional profiling of shear-exposed cultured endothelial cells commonly identifies eNOS as a gene whose steady-state expression is regulated by shear.28–30 Moreover, eNOS is 1 example of only a small percentage (
1% to 5%) of endothelial genes that are regulated by prolonged exposure to laminar shear stress.31 Importantly, in vitro culture models have provided strong evidence that the eNOS gene is regulated transcriptionally by shear stress. The transcription factors Krüppel-like factor-2 (KLF2) and nuclear factor 
B have both been implicated in this transcriptional response. Electromobility shift assays, promoter deletions, and promoter mutations suggest that nuclear factor B subunits p50 and p65 interact functionally with a 5'-GAGACC-3' sequence located upstream of the eNOS transcription start site and are key for shear activation of the eNOS promoter.32 KLF2 expression is induced by shear stress in vascular endothelium, can induce transcriptional activation of the eNOS promoter, and functionally interacts with cis-regulatory regions located upstream of the eNOS transcription start site; interestingly, the expression of KLF2 has been addressed in the developing chick embryo, revealing an overlapping expression pattern of KLF2 and eNOS in the embryonic cardiovascular system.33 We recently demonstrated that the transcription of eNOS was reduced in regions of the adult mouse aorta that exhibit complex or disturbed laminar flow and are predisposed to atherosclerosis.18 A similar expression pattern was also observed in mouse strains that were either susceptible or resistant to atherosclerosis. We also demonstrated that regulation of transcription contributed to increased eNOS expression in response to shear stress in vitro.18 Future studies will be helpful in defining the important cis- and trans-pathways in the development-associated induction of eNOS transcription at E9.5, as well as the later decrease in expression from E11.5 to E14.5.
Transcription of the eNOS gene was not restricted to the cells of the cardiovascular system during mammalian development. Our demonstration that the eNOS gene is transcriptionally active and that the native mRNA and protein are present in columnar cells of the AER is of significance. The AER is a thickening of the ectoderm at the most distal edge of the limb bud that directs mesenchymal outgrowth of the limb bud through modulation of the underlying progress zone.20 To date, the signaling molecule(s) used by the AER to regulate growth of limbs and digits has not been conclusively identified, although strong modulatory roles for retinoic acid and fibroblast growth factor are suspected.20,34 Elf-1 is an Ets family member that was previously shown to be a component of the nucleoprotein complexes forming on one of 2 positive regulatory domains within the human eNOS promoter.1 A role for Elf-1 has emerged in the regulation of vascular-specific gene expression during blood vessel development35 and in developing limb buds. It is tempting to speculate that eNOS-derived NO is an additional signaling molecule in the development of the AER.
We observed eNOS expression in other regions of the developing limbs. We demonstrated that the eNOS promoter is active in vitro in embryonic myoblasts and in vivo in proximal skeletal tissue during myogenesis. This phase of skeletal muscle cell development corresponds to secondary myogenesis, when a second wave of myoblasts emerges from the proximal somitic regions and aligns with primary skeletal myotubes at E14.5 to E17.5. NO has been implicated in the fusion of myoblasts to form myotubes.36 Vascular cell adhesion molecule-1 is another relatively endothelial cell-restricted molecule that is uniquely expressed at this embryonic stage of skeletal muscle differentiation. Vascular cell adhesion molecule-1 is important in myogenesis, specifically in the transition of secondary myoblasts to secondary myotubes.37–39
Limb reduction defects have been described as a variably penetrant phenotype in eNOS–/– mice.40 This developmental defect has previously been attributed to aberrant angiogenesis and/or impaired blood flow to the developing limb. The present work suggests that the limb growth abnormalities in these eNOS–/– mice represent functional defects in NO, derived either from the AER and/or the proximal myocytes. Consistent with this notion, in Drosophila, NO is known to function as a signaling molecule in limb morphogenesis by creating a patterning gradient.41 This newer role for eNOS-derived NO during limb patterning during development may have therapeutic relevance to our understanding of muscle repair42 and provide insight into the induction of eNOS expression in diseases of adult skeletal muscle.
Although we focused the present work on embryonic expression of eNOS, recent work from others has highlighted expression of eNOS in extraembryonic tissues. Nath et al documented expression of eNOS in the endothelium of extraembryonic blood vessels, presumably the vitelline circulation of the yolk sac, especially at E8.5.43 We also observed strong eNOS promoter/β-galactosidase reporter expression in the yolk sac, especially the endothelial layer of the yolk sac, which is consistent with the findings of others.43,44 This is not surprising given that all 3 NOS isoforms are present in preimplantation embryos.45,46 In the embryo, we argue that the physical forces of the circulation are important to the expression of eNOS in the developing embryonic vasculature. Flow may also be relevant to the extraembryonic expression of eNOS. In this respect, flow in the vitelline circulation is elevated earlier, or to a greater extent, than in the embryo at early stages of life. Mu and Adamson47 used ultrasound biomicroscopy to noninvasively image and record Doppler blood velocity waveforms during development. Robust directional flow was evident in the vitelline artery to the yolk sac at E8.5. The flow through the vitelline circulation is clearly more robust relative to the developing embryo at E8.5 versus E9.5.47 Whether this could account for the earlier onset of eNOS expression in extraembryonic tissues remains to be defined. We also cannot exclude that laminar versus disturbed flow could account for some of the observed differences in eNOS expression between yolk sac blood vessels and blood vessels of the embryo proper.18
In summary, in the present work, we have presented a comprehensive examination of the expression of the eNOS gene during mammalian embryogenesis, focusing principally on cardiovascular development. We demonstrated unique spatial and kinetic transcription expression of the eNOS gene that followed the establishment of embryonic blood flow in the developing mammal. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and in the AER of developing limbs, suggesting a role for eNOS-derived NO in limb development.
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Acknowledgments |
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Sources of Funding
A.M.T. is the recipient of a Canadian Institute for Health Research doctoral research award. Y.-Q.Z. is the recipient of a fellowship from the Ontario Research and Development Challenge Fund. J.A.S. is the recipient of a Canadian Institute for Health Research postdoctoral fellowship. The ultrasound biomicroscope was purchased with funding from the Richard Ivey Foundation. P.A.M. and S.L.A. are supported by Canadian Institute for Health Research operating grants (MOP 37778 and MOP 12772). P.A.M. is the recipient of a Career Investigator Award from the Heart and Stroke Foundation of Canada.
Disclosures
None.
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Footnotes |
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Original received February 23, 2005; resubmission received February 7, 2008; revised resubmission received May 25, 2008; accepted May 29, 2008.
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