Mature Vascular Endothelium Can Give Rise to Smooth Muscle Cells via Endothelial-Mesenchymal Transdifferentiation
In Vitro Analysis
Though in the past believed to be a rare phenomenon, endothelial-mesenchymal transdifferentiation has been described with increasing frequency in recent years. It is believed to be important in embryonic vascular development, yet less is known regarding its role in the adult vasculature. Using FACS and immunomagnetic (Dynabeads) purification techniques (based on uptake of DiI-acetylated low-density lipoproteins and/or PECAM-1 expression) and double-label indirect immunostaining (for endothelial and smooth muscle [SM] markers), we demonstrate that mature bovine vascular endothelium contains cells of an endothelial phenotype (defined by VE-cadherin, von Willebrand factor, PECAM-1, and elevated uptake of acetylated low-density lipoproteins) that can undergo endothelial-mesenchymal transdifferentiation and further differentiate into SM cells (as defined by expression of α-SM-actin, SM22α, calponin, and SM-myosin). “Transitional” cells, coexpressing both endothelial markers and α-SM-actin, were consistently observed. The percentage of cells capable of endothelial-mesenchymal transdifferentiation within primary endothelial cultures was estimated as 0.01% to 0.03%. Acquisition of a SM phenotype occurred even in the absence of proliferation, in γ-irradiated (30 Gy) and/or mitomycin C–treated primary cell cultures. Initiation of transdifferentiation correlated with disruption of cell-cell contacts (marked by loss of VE-cadherin expression) within endothelial monolayers, as well as with the action of transforming growth factor-β1. In conclusion, our in vitro data show that mature bovine systemic and pulmonary endothelium contains cells that can acquire a SM phenotype via a transdifferentiation process that is transforming growth factor-β1– and cell-cell contact–dependent, but proliferation-independent.
Until recently, endothelial cells (ECs) and smooth muscle cells (SMCs) were believed to arise from separate precursor cells. However, experiments in a murine embryonic stem cell model demonstrated that a common precursor cell can differentiate toward either endothelial or SM lineage.1 Furthermore, a progenitor cell at different steps of a given differentiation pathway can switch its phenotype, a phenomenon called transdifferentiation. Studies of embryonic avian aortic and human pulmonary vascular development demonstrated that the endothelium itself could be the source of at least some SMCs in the arteries.2–7⇓⇓⇓⇓⇓ However, whether transdifferentiation of ECs into SMCs can occur in adult vasculature is not clear. Several in vivo and in vitro studies have demonstrated that, in the adult, ECs can transform at least into mesenchymal cells.8–10⇓⇓ Yet, a more advanced differentiation of endothelial-derived mesenchymal cells into SMCs has, to our knowledge, not been described.
We hypothesized that endothelial-mesenchymal transdifferentiation can take place in the adult vasculature, and that endothelium-derived mesenchymal cells could further differentiate toward a SM phenotype. Utilizing an in vitro approach, we demonstrated that alterations in endothelial phenotype, indicative of transdifferentiation toward a SM phenotype, occurred in the purified primary cultures of bovine aortic and/or pulmonary artery endothelium. We then estimated the time course of this process and sought mechanisms by which this phenomenon is controlled, including a role of proliferation, transforming growth factor-β1 (TGF-β1), and integrity of cell-cell contacts. Our data demonstrate that mature bovine vascular endothelium contains cells that, in vitro, can acquire a SMC phenotype via a transdifferentiation process that is TGF-β1– and cell-cell contact–dependent, but proliferation-independent. The observation that endothelial-smooth muscle transdifferentiation can take place in primary cell cultures derived from the adult vasculature suggests that endothelium could serve as a potential source of at least some SMCs in vascular neointimal lesions.
Materials and Methods
Isolation of Primary EC Cultures
Endothelium from adult bovine aortas and/or main pulmonary arteries (n=37) was isolated as intact monolayers, using brief pretreatment with Dispase (Becton Dickinson) followed by a single scrape with a flexible plastic scraper (Nalge/NUNC). Cell monolayers were plated onto at least 10 culture dishes (33 mm, diameter), either plastic or covered with 1% denatured type I collagen (gelatin, Sigma Chemical) in complete DMEM (Sigma) with 10% calf serum (HyClone Laboratories). Primary cultures were confirmed to be free of contamination with SMCs by thorough screening of at least 2 culture dishes subjected to immunostaining for SM markers. Contamination with subendothelial nonmuscle cells11 was ruled out based on the following: first, when subendothelial cells are isolated in primary culture, they appear as individual spindle-shaped cells sparsely incorporated within extracellular matrix (Figure 3B), in contrast to morphologically cobblestone endothelium isolated as monolayers (Figure 3A). Second, nonmuscle cells in subendothelial layer of bovine arteries do not express EC markers employed in this study (not shown).
Antibodies and Cytokines
The following antibodies (Abs) were used: monoclonal antibodies (mAbs) against α-SM-actin (clone 1A4) (1:100, Sigma), rabbit anti–von Willebrand factor Abs (1:100, DAKO), goat anti–vascular endothelial (VE)–cadherin, and rabbit anti–platelet-endothelial cell adhesion molecule-1 (PECAM-1) (CD-31) Abs (1:10, Santa Cruz Biotechnology). Anti-SM22α mAbs (clone 1B8) were a generous gift of Dr S. Sartore (University of Padua, Italy). mAbs against SM myosin heavy chains (SM-MHCs) and calponin were previously described.11,12⇓ Human recombinant TGF-β1 and neutralizing chicken Abs against active TGF-β1 were from R&D Systems (No. AF-101-NA, lot No. FS08). Neutralizing TGF-β1 Abs were added every day in fresh medium.
Fluorescence-Activated Cell Sorting (FACS) and Immunomagnetic (Dynabeads) Purification Techniques
Primary EC cultures were metabolically labeled with DiI-acetylated low-density lipoproteins (DiI-Ac-LDL) (Biomedical Technologies) for 4 hours at 10 μg/mL or for 16 hours at 5 μg/mL13 and subjected to FACS analysis (sorter model Moflow by Cytomation). Cell fraction exhibiting elevated uptake of DiI-Ac-LDL was re-plated in culture and subsequently re-sorted by FACS. As negative controls, bovine SMCs and adventitial fibroblasts were used and have been confirmed (as described elsewhere13) to be incapable of metabolizing Ac-LDL (not shown).
For FACS-purification based on PECAM-1 expression, primary (d1) ECs were dispersed with Accutase (Innovative Cell Technologies) and split in 2 portions to incubate one with anti–PECAM-1 antibodies and another with nonimmune rabbit IgG as a negative control. After 2-hour incubation on ice, gently shaking, cells were incubated with anti-rabbit IgG conjugated with Alexa-488 fluorescent dye (1:250, Molecular Probes) and subjected to FACS analysis. Cell fraction positive for PECAM-1 expression (Figure 1, F-2) was re-plated in culture. Purification of primary (d1) ECs was also performed using superparamagnetic polystyrene beads (Dynabeads) conjugated with anti–PECAM-1 mAbs (Dynal Biotech) as described elsewhere.14
Cells were fixed in 1% paraformaldehyde (1 minute), followed by absolute methanol (−20°C, 10 minutes) and incubated with primary antibodies for 1 hour at room temperature. Secondary antibodies were either conjugated to Alexa-488 fluorescence dye (Molecular Probes) or to biotin (DAKO). For the latter, streptavidin-Alexa-594 (Molecular Probes) was used. Stained cells were embedded in VectaShield mounting medium with DAPI (Vector Laboratories) and examined with a Nikon Optiphot epifluorescence microscope with the DEI-470 Optronics imaging system.
Inhibition of Mitogenesis
Gamma (γ)-irradiation (30 Gy from a cesium source) and mitomycin C treatment (2 μg/mL, 2 hours) were used to inhibit cell replication in primary EC cultures maintained in DMEM with 10% serum.
Primary EC Cultures Give Rise to SMCs
Primary (day 1) arterial EC cultures were first confirmed to be free of contamination with SMCs and/or subendothelial nonmuscle cells (see Materials and Methods). Cultures were further purified by FACS and/or immunomagnetic (Dynabeads) techniques based on 2 endothelial-specific markers: elevated uptake of DiI-Ac-LDL (FACS-based purification) and/or PECAM-1 expression (both FACS and Dynabeads immunopurification). In primary endothelial cultures metabolically labeled with DiI-Ac-LDL, the cell fraction exhibiting elevated DiI-Ac-LDL uptake was selected by FACS (Figure 1A), re-plated onto several dishes, and subsequently reassessed for DiI-Ac-LDL uptake at different days in culture. As shown in Figure 1, an EC fraction, selected as positive for high DiI-Ac-LDL uptake (Figure 1A), over time gave rise to cells incapable of metabolizing DiI-Ac-LDL (Figures 1B through 1E). Based on sequential FACS analysis, these latter cells were estimated to comprise approximately 0.01% to 0.03% of the total number of ECs on day 3 in culture (Figure 1B), 0.09% on day 5 (not shown), 1.46% on day 11 (Figure 1C), and 8.95% on day 17 (Figure 1D). By the second passage in subculture, they constituted approximately 44% of the total number of cells (Figure 1E). The cells incapable of DiI-Ac-LDL uptake appeared with time in primary cultures as very elongated, spindle-shaped cells forming a network on top of the EC monolayer (Figure 1G) and, with time, piling on each other. Colonies of these mesenchymal-like cells were selectively isolated using Teflon cloning rings, as well as by FACS based on negative DiI-Ac-LDL uptake, and assessed for expression of SM markers by indirect immunofluorescence analysis. These data demonstrated a progressive mesenchymal-to-smooth muscle differentiation of these cells in culture. During the first days after isolation, cells maintained their spindle morphology and weakly expressed α-SM-actin in a diffuse, cytoplasmic pattern (Figure 2A). With time in culture, cells began to express SM22α, calponin, and, in some cells, SM-MHCs, all in a diffuse cytoplasmic pattern (Figures 2C, 2E, and 2G). Progressively, the cells became larger, and all 4 SM proteins were expressed in characteristic stress-fiber patterns (Figures 2B, 2D, 2F, and 2H). At this time, no endothelial-related markers were detected.
Importantly, if primary EC cultures were allowed to expand for more than 5 days and only then subjected to FACS based on DiI-Ac-LDL uptake, the postsorted DiI-Ac-LDL–positive EC fraction did not yield any mesenchymal cells. This suggests that the process of endothelial-mesenchymal transdifferentiation is initiated during first days after isolation of endothelial monolayers in culture.
In addition, purification of primary EC cultures was performed using another endothelial-specific marker, PECAM-1, by both FACS and immunomagnetic (Dynabeads) sorting techniques. As stated, before utilizing these techniques, primary EC cultures were confirmed to be free of contamination with non-ECs by other methods. After FACS and/or Dynabeads sorting based on PECAM-1 expression, the obtained EC fractions were evaluated over time in culture. The results were similar to those obtained with DiI-Ac-LDL FACS analysis. That is, primary ECs, selected on day 1 for PECAM-1 expression by either FACS (Figure 1F) or Dynabeads (not shown), over time yielded mesenchymal cells, which further differentiated into SMCs (similar to that shown in Figure 2). Again, as mentioned, if EC purification based on PECAM-1 expression was performed after day 5 in primary culture, no mesenchymal or SMCs were observed.
Time Course of Acquisition of a SM Phenotype in Primary EC Cultures
Endothelium was isolated as “intact” cell monolayers (Figure 3A and 3C), which were plated onto plastic culture dishes. Endothelial monolayers attached within 18 to 24 hours (Figure 3D) and acquired a typical cobblestone appearance. ECs within monolayers were positive for all endothelial markers tested (VE-cadherin, PECAM-1, von Willebrand factor, high uptake of Ac-LDL) (not shown). No cells expressing SM markers (α-SM-actin, SM22α, calponin, SM-MHCs) were detected on day 1 in culture (Figure 3E, right panel). The expression of EC and SMC markers in these cultures was assessed daily.
On day 1 after isolation, cell-cell contacts in endothelial monolayers appeared tight and were defined by vivid expression of VE-cadherin (Figure 3E, left panel). However, by day 2, cell-cell contacts became disrupted (Figure 3F, left panel), and cells were observed that expressed α-SM-actin in a diffuse, cytoplasmic pattern (Figures 3F and 3H, right panels). Notably, these cells still exhibited an epithelioid morphology similar to other ECs within the monolayer. Some cells coexpressed α-SM-actin and endothelial markers (von Willebrand factor antigen, VE-cadherin, and increased uptake of Ac-LDL) (Figure 3F, VE-cadherin, others not shown). By day 4 in culture, more ECs expressed α-SM-actin (Figure 3I). By days 5 and 6, endothelial monolayers were no longer compact, especially at the monolayer border (Figure 4A), where many ECs had lost their rounded appearance and acquired an elongated, mesenchymal-like morphology (Figures 3J, 4A, and 4⇓B). These cells no longer expressed VE-cadherin (Figure 4B), and some of them now expressed α-SM-actin in a diffuse, cytoplasmic pattern (Figures 3J and 4⇓C). Expression of SM-MHCs was not noted at that time. By days 11 to 14, α-SM-actin was expressed in a microfilamentous, stress-fiber pattern, and some α-SM-actin–positive cells now expressed SM-MHCs in a diffuse cytoplasmic pattern (similar to that shown in Figure 2D).
Phenotypic Differences Between EC Cultures Plated Onto Plastic and/or Denatured Collagen
When the freshly isolated endothelial monolayers were plated onto denatured collagen, rather than onto plastic, marked differences were observed with regard to time-related changes in cell morphology and expression of endothelial and SM markers. In contrast to endothelial monolayers plated onto plastic (described in “Time Course of Acquisition…,” Figures 4A through 4C), those plated onto denatured collagen maintained over time a more compact appearance (Figure 4D) and more defined cell-cell contacts (Figure 4E). In these cultures (also negative for SM marker expression on day 1), the initial process of α-SM-actin acquisition appeared similar to that in cultures plated on plastic. That is, during the first 2 to 3 days in culture, some cells within the monolayers began to express α-SM-actin in a cytoplasmic pattern (not shown). Again, certain cells coexpressing α-SM-actin and VE-cadherin were observed. However, approximately by day 5, cell monolayers had reestablished their cobblestone appearance with tight cell-cell contacts (Figure 4E), and very few α-SM-actin-positive cells were detected at that time. Over time, endothelial monolayers grown on denatured collagen did not yield spindle-shaped cells expressing α-SM-actin.
Acquisition of a SM Phenotype Occurs in the Absence of Proliferation
To test whether the acquisition of a SM phenotype by select ECs within the endothelial monolayers required DNA synthesis, day 1 EC cultures, maintained in DMEM with 10% serum, were either γ-irradiated (30 Gy) or treated with mitomycin C. Inhibition of DNA synthesis was confirmed by the absence of nuclear BrdU uptake (not shown). These endothelial cultures, initially negative for α-SM-actin expression, by day 4 yielded cells that expressed α-SM-actin in a diffuse cytoplasmic pattern (Figure 5B). By day 7, cells were observed that exhibited an elongated, often stellate morphology and expressed α-SM-actin in a microfilamentous stress-fiber pattern (Figure 5C). Cells that coexpressed both endothelial and SM-related markers (von Willebrand factor [vWf] and α-SM-actin, respectively) were routinely detected by double immunofluorescence labeling (Figures 5D through 5G). Some cells positive for α-SM-actin concurrently expressed SM-MHCs (not shown); however, no cells were observed to concurrently express vWf and SM-MHCs.
TGF-β Is Involved in Inducing Endothelial-Mesenchymal Transdifferentiation
We used a dual approach to test the hypothesis that TGF-β1 may play an important role in inducing endothelial-mesenchymal transdifferentiation in our system. First, primary EC cultures, plated onto denatured collagen (in which case acquisition of SM phenotype was not normally observed), were treated with TGF-β1 (0.25 to 1 ng/mL). Second, primary EC cultures, plated onto plastic (in which case acquisition of SM phenotype was normally observed), were treated with neutralizing antibodies against active TGF-β1.
In the first set of experiments, treatment of endothelial monolayers plated on denatured collagen with TGF-β1 resulted in profound disruption of cell-cell contacts. Cells acquired a spindle-shaped morphology and, within the first week in culture, some of them began to express α-SM-actin, SM22α, calponin, and SM-MHCs (not shown). Control primary EC cultures, grown on denatured collagen, maintained a compact cobblestone appearance and did not yield SMCs over time. As an additional control, a commercially available CPAE endothelial cell line (American Type Culture Collection, Manassas, Va), as well as ECs derived by late (after day 5 in primary culture) FACS purification were treated with 0.25 to 1 ng/mL of TGF-β1. In these cell cultures, α-SM-actin was detected in some cells, whereas SM-MHC expression was not detected even after a 20-day treatment (not shown; other SM proteins were not assessed).
In a second set of experiments, ECs plated onto plastic were incubated with neutralizing TGF-β1 antibodies (Abs) (5 and 10 μg/mL). As expected, untreated endothelial monolayers grown on plastic over time gave rise to spindle-shaped cells expressing SM proteins in a stress-fiber pattern (Figure 6A, α-SM-actin is shown). In cultures treated with neutralizing TGF-β1 Abs at 5 μg/mL, α-SM-actin–positive cells were still observed, yet α-SM-actin was expressed in a cytoplasmic pattern, and cells maintained an epithelioid morphology over time (Figure 6B). Transition to spindle-shaped morphology with a stress-fiber pattern of α-SM-actin did not occur, and SM-MHC expression was not detected in these cultures (SM22α and calponin were not assessed). In endothelial monolayers treated with 10 μg/mL of neutralizing Abs, no α-SM-actin–positive cells were observed over time (Figure 6C). Similar strategy was used for γ-irradiated (30 Gy) primary EC cultures (data not shown). Treatment with neutralizing Abs (10 μg/mL) suppressed appearance of α-SM-actin–positive cells, whereas untreated γ-irradiated primary cultures yielded α-SM-actin–positive cells similar to that described (not shown).
Endothelial-to-mesenchymal transdifferentiation is increasingly being viewed as an important biological process in development and disease. Studies of embryonic avian aortic and human pulmonary vascular development have demonstrated that vascular endothelial cells could undergo an endothelial-mesenchymal phenotypic switch and migrate into the subendothelial media, suggesting that at least some SMCs in the embryonic arteries arise from the endothelium.2–7⇓⇓⇓⇓⇓ Such phenotypic “plasticity” could be maintained by at least some ECs in the neonatal period, as recently demonstrated in human umbilical vein ECs (HUVECs) that were shown capable of transdifferentiating into beating cardiomyocytes.15 Whether any cells within the mature vascular endothelium retain this capability has remained unclear. Interestingly, this possibility was suggested nearly 60 years ago by R. Altschul16 who, in histological studies of human atherosclerosis, described that some aortic ECs underwent an endothelial-mesenchymal phenotypic switch and invaded the subendothelial space. This concept was recently revived in speculations that endothelial-derived mesenchymal cells may be involved in development of neointimal lesions in transplant atherosclerosis and restenosis.17–19⇓⇓ In addition, transdifferentiation of ECs into mesenchymal cells in the adult is suggested by findings in experimental wound repair where capillary ECs converted into interstitial connective tissue cells in granulation tissue and by findings that microvascular ECs transdifferentiated into mesenchymal spindle-shaped cells on chronic inflammatory stimuli.8,20–23⇓⇓⇓⇓ Further, in mature ECs, the capacity for transdifferentiation is supported by findings that adult bovine aortic ECs, when treated with TGF-β1, convert to spindle-shaped cells expressing α-SM actin,9 and that certain murine endothelial-like cell lines irreversibly transform into mesenchymal cells on overgrowth in culture.10 Our findings complement and extend these previous observations and provide rigorous in vitro evidence that mature vascular endothelium contains cells that have the potential to undergo endothelial–smooth muscle transdifferentiation.
An important and novel observation of the present study was that primary EC cultures confirmed to be free of contamination with non-ECs and further purified by FACS and/or magnetic beads (Dynabeads) techniques (based on elevated uptake of DiI-Ac-LDL and/or PECAM-1 expression) gave rise to mesenchymal and even SM cells. Both FACS and Dynabeads purifications were performed soon after initiating primary culture, ie, on day 1 after EC isolation. When the cell sorting was performed on day 5 after cell isolation or later, no mesenchymal cells were observed to appear in these cultures because the endothelial-mesenchymal phenotypic switch had probably already occurred and the mesenchymal cells had been selectively sorted out. FACS and Dynabeads sorting techniques are commonly used by investigators to obtain pure endothelial cultures (ie, to specifically eliminate mesenchymal “contaminant”); however, they are usually applied after cells expand in culture for some time. In studies of microvascular endothelial cells, sorting techniques are often applied at the time of cell isolation; however, the need for subsequent sorting is noted as an essential requirement for maintaining purity of EC cultures.13,14,24⇓⇓ This is an interesting detail because, for example, dermal microvascular ECs are described as very susceptible to transdifferentiation into mesenchymal cells.21 Thus, our data would support the assumption that the mesenchymal cells, often observed in primary EC cultures, are not the result of contamination but are often the result of transdifferentiation.
Additional experimental evidence supports our contention that the appearance of SM-like cells in primary endothelial cultures is due to transdifferentiation. First, the primary endothelial monolayers were used for analysis, which displayed typical cobblestone morphology and were composed of endothelial cells only (see Results). Several endothelial-specific markers were expressed by these cells, specifically, VE-cadherin was expressed by all cells within the monolayer (Figure 3E, left). No staining for SM-related markers was detected at day 1 after isolation. The presence of subendothelial nonmuscle cells was also ruled out (these cells, isolated as controls by a deeper scrape, appeared as individual cells of spindle-shaped morphology sparsely incorporated within the extracellular matrix (Figure 3B) and did not express endothelial markers (not shown). Furthermore, “transitional” cells coexpressing both endothelial (VE-cadherin, von Willebrand factor) and SM-related (α-SM-actin) markers were consistently observed. Endothelial-mesenchymal phenotypic switch was accompanied by changes in cell shape toward mesenchymal, spindle-shaped, which continued to evolve as the cells expressed SM phenotype. Finally, the transdifferentiation process could be experimentally manipulated. It was attenuated by maintaining primary ECs on denatured collagen, which could be reversed by addition of TGF-β1. Moreover, the process was facilitated by plating primary EC cultures on plastic, which was inhibited by addition of neutralizing TGF-β1 antibodies. Importantly, studies in epithelial cells have pointed out the requirement for TGF-β for a change in the matrix environment, for a breakdown of E-cadherin expression with subsequent alteration of cell morphology, and for increased expression of α-SM-actin.25–32⇓⇓⇓⇓⇓⇓⇓ Other studies have emphasized that myogenesis is controlled by the cell’s shape.33 Thus, it seems likely that endothelial- and epithelial-to-mesenchymal transdifferentiation processes share many common molecular mechanisms.
Our findings are the first, to our knowledge, to describe the acquisition of a more differentiated SM phenotype (defined by expression of SM-MHCs rather than just that of α-SM-actin) in the course of endothelial–smooth muscle transdifferentiation in the cells from mature vessels. The data of the present study suggest a critical role for TGF-β1 in the transdifferentiation of ECs. Although TGF-β1 is known to promote α-SM-actin expression in nonmuscle cells (ECs and fibroblasts derived from various tissues9,34⇓), it is currently thought not to be sufficient to induce expression of late SM differentiation markers, such as SM-MHCs, in cells of non-SMC lineage.34 Thus, it is possible that in our system TGF-β1 is necessary for initiation of transdifferentiation, yet it may not be sufficient for driving the process to the higher SM differentiation level described (defined by expression of SM-MHCs). If so, the other factors/mechanisms operating to cause transdifferentiation of ECs into differentiated SMCs need to be identified.
Because only a small percentage (0.01% to 0.03%) of cells within the endothelium appear capable of undergoing endothelial–smooth muscle transdifferentiation, questions arise regarding the origin of these cells. In the adult, bone marrow–derived progenitor cells from circulating blood can incorporate into endothelial monolayer, differentiate into ECs, and constitute a significant proportion of cells within the vascular endothelium.35,36⇓ Furthermore, bone marrow–derived progenitor cells have been described that have the potential to differentiate into endothelial/mesothelial and SM-like cells.37,38⇓ Recently, Kaushal et al39 have demonstrated that acellular vascular grafts seeded with blood-borne endothelial progenitor cells later become populated with contractile SMCs, thus raising a possibility of gradual transformation of endothelial progenitor cells to vascular ECs to medial SMCs. Taken together, these observations and our data raise the possibility that endothelial-like cells within the mature endothelium, which are capable of transdifferentiating into SMCs, may have originated from a circulating pool of bone marrow–derived endothelial precursors. If this were the case, mature vascular endothelium, having a constant supply of blood-borne multipotent endothelial-like cells, could itself serve as a potential source of at least some SM-like cells in vascular neointimal lesions.
This study was supported by NIH grants SCOR HL-57144, PPG HL-14985. The authors gratefully acknowledge Stephen Hofmeister for technical assistance, Dr John T. Reeves for valuable discussion, and Marcia McGowan for help in preparation of the manuscript.
Original received November 7, 2001; resubmission received March 15, 2002; revised resubmission received April 29, 2002; accepted April 29, 2002.
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