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agonists inhibit TGF-
induced pulmonary myofibroblast differentiation and collagen production: implications for therapy of lung fibrosis
Departments of 1Environmental Medicine, 2Medicine, 3Pediatrics, and 4Lung Biology and Disease Program, and 5the Cancer Center, University of Rochester School of Medicine, Rochester, New York
Submitted 12 October 2004 ; accepted in final form 14 February 2005
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ABSTRACT |
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is a transcription factor that upon ligation with PPAR agonists activates target genes containing PPAR response elements. PPAR agonists have anti-inflammatory activities and may have potential as antifibrotic agents. In this study, we examined the abilities of PPAR agonists to block two of the most important profibrotic activities of TGF-
on pulmonary fibroblasts: myofibroblast differentiation and production of excess collagen. Both natural (15d-PGJ2) and synthetic (ciglitazone and rosiglitazone) PPAR agonists inhibited TGF--driven myofibroblast differentiation, as determined by 
-smooth muscle actin-specific immunocytochemistry and Western blot analysis. PPAR agonists also potently attenuated TGF--driven type I collagen protein production. A dominant-negative PPAR partially reversed the inhibition of myofibroblast differentiation by 15d-PGJ2 and rosiglitazone, but the irreversible PPAR antagonist GW-9662 did not, suggesting that the antifibrotic effects of the PPAR agonists are mediated through both PPAR-dependent and independent mechanisms. Thus PPAR agonists have novel and potent antifibrotic effects in human lung fibroblasts and may have potential for therapy of fibrotic diseases in the lung and other tissues.
peroxisome proliferator-activated receptor-; myofibroblasts; 15d-PGJ2; ciglitazone; rosiglitazone
is a profibrotic cytokine that is a key molecule in the development of pulmonary fibrosis. Its expression is increased in many patients with fibrotic lung diseases (1, 2, 4). Furthermore, overexpression of TGF- in the lung causes severe and irreversible pulmonary fibrosis (31). Inhibiting the effects of TGF- in vivo abrogates the development and progression of pulmonary fibrosis (2, 23, 24).
The fibroblast is now recognized as the key effector cell in the normal wound-healing process, as well as the development of fibrosis (34). Fibroblasts can transdifferentiate to myofibroblasts following exposure to a variety of stimuli, particularly TGF- (29, 33, 34). Myofibroblasts are fibroblast-like cells with smooth muscle cell characteristics, particularly expression of -smooth muscle actin (-SMA). They are one of the major sources of extracellular matrix proteins, especially collagen, as well as fibrogenic cytokines and chemokines. The accumulation of fibroblasts and myofibroblasts and their production of extracellular matrix proteins, e.g., collagen, result in significant damage to the lung architecture and gas exchange abnormalities (29, 30, 34, 38). During normal wound repair, myofibroblasts undergo apoptosis and are removed from the healing area. However, in progressive fibrosis, myofibroblasts fail to apoptose and can persist within the fibrotic scar, perpetuating the scarring process (29, 38). A potential novel and exciting therapy to inhibit scarring is to prevent the conversion of fibroblasts to myofibroblasts and/or to decrease myofibroblast production of collagen and other extracellular matrix proteins. This can be studied in vitro by a model system using TGF- to drive the differentiation of cultured primary human pulmonary fibroblasts to myofibroblasts (33).
Peroxisome proliferator-activated receptor (PPAR)- is a nuclear receptor. After ligation with its agonist, PPAR heterodimerizes with the retinoid X receptor (RXR). This complex recognizes PPAR response elements (PPRE) in promoters on target genes resulting in the regulation of gene transcription (3, 5, 36). PPAR agonists have novel effects on fibroblast differentiation by promoting their conversion to adipocytes (13, 21). Over the past decade PPAR agonists have received attention for their ability to regulate adipocyte differentiation and to increase insulin sensitivity in diabetic patients (8, 25, 36). 15-Deoxy-
12,14-prostaglandin J2 (15d-PGJ2) is a potent naturally occurring PPAR agonist. It is a spontaneously derived end product from prostaglandin D2 (3, 17). A variety of synthetic PPAR agonists have now been developed for the treatment of diabetes. These drugs include the thiazolidinediones (TZDs): trioglitazone, rosiglitazone, ciglitazone, and pioglitazone (8, 25, 37). Rosiglitazone is the one of the most specific TZDs for PPAR (8, 25) and is currently in clinical use as Avandia (GlaxoSmithKline) for therapy of Type II diabetes.
There are important new indications that PPAR agonists have a role in modulating inflammation (5 –7, 36). PPAR has anti-inflammatory activities, including the repression of the NF-
B and activator protein (AP)-1 pathways (5, 6, 10). We hypothesize that PPAR agonists have antifibrotic properties by inhibiting pulmonary myofibroblast differentiation and collagen production. In the current study we provide novel data demonstrating the ability of both endogenous and synthetic PPAR agonists to block key TGF--mediated profibrotic effects, including pulmonary myofibroblast differentiation and excess collagen production. These activities support the concept that PPAR agonists may have exciting potential for therapy of currently untreatable fibrotic lung diseases.
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METHODS |
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agonists ciglitazone and 15d-PGJ2 (Biomol, Plymouth Meeting, PA) and rosiglitazone (Cayman Chemical, Ann Arbor, MI) were prepared as 10 mM stocks in DMSO and added to cell cultures to the final concentrations indicated. WY-14643 (Biomol), a PPAR agonist, and GW-9662 (Cayman Chemical), an irreversible PPAR antagonist, were prepared in the same manner. DMSO was added to negative control wells at a final concentration of 0.1%. Recombinant human TGF-1 was purchased from R&D Systems (Minneapolis, MN). A monoclonal -SMA antibody (Sigma Aldrich) was used for both Western blots and immunocytochemistry (ICC). Western blots were normalized to GAPDH (Abcam, Cambridge, MA). The PPAR antibody recognizes PPAR1 and 2 (Calbiochem); the RXR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) recognizes RXR, , and and does not cross-react with retinoic acid receptor (RAR).
Detection of PPAR and RXR protein.
Five normal primary human pulmonary fibroblast cultures from different patients were analyzed for PPAR and RXR protein expression. Lysates containing 10 µg of protein and 500 ng of human adipose tissue as a control were examined by Western blot for expression of PPAR protein with a polyclonal mouse antibody (Calbiochem) and horseradish peroxidase (HRP) goat anti-mouse antibody (Jackson Immunoresearch, West Grove, PA). The membrane was stripped and reprobed with an antibody that recognizes all three forms of RXR protein expression (Santa Cruz) and HRP goat anti-mouse antibody (Jackson Immunoresearch).
ICC for -SMA.
Normal primary human pulmonary fibroblasts were plated at a density of 2,000 cells per well on four-well chamber slides (Nalge Nunc International, Naperville, IL) in MEM supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 units/ml), streptomycin (100 µg/ml), and amphotericin (0.25 µg/ml, Life Technologies) at 37°C in 7% CO2. Fibroblasts were treated for 72 h with either human recombinant TGF- (R&D Systems) at 5 ng/ml alone or with one of the following agonists: 15d-PGJ2 (10 µM), ciglitazone (25 µM), and rosiglitazone (10 and 20 µM). WY-14643, a PPAR agonist (25 µM), and DMSO (0.1%) were included as negative controls. Cells were then fixed with methanol and sequentially treated with mouse monoclonal -SMA antibody (Sigma) or IgG2A isotype control (R&D Systems), biotinylated goat anti-mouse (Vector Labs), and HRP-streptavidin conjugate (Jackson Immunoresearch). Cells were then stained with aminoethyl carbazole (Zymed) and counterstained with Gill's hematoxylin no. 1 (Sigma) before mounting with Immunomount (Shandon).
Western blot for -SMA.
Primary human pulmonary fibroblasts were plated in duplicate with 100,000 cells/well in six-well plates (Corning, Corning, NY). Cells received the same treatments as cells prepared for ICC. Lysates containing 2 µg of protein were separated on a 12% SDS gel in reducing conditions and were examined for expression of -SMA protein and visualized by Western Lightning Chemiluminescence Reagent Plus kit (Perkin-Elmer, Wellesley, MA). Densitometry of the resulting bands was performed using Kodak 1D Image Analysis Software (Rochester, NY).
Type I collagen ELISA.
Human pulmonary fibroblasts were grown in 12-well plates and treated in quadruplicate with either media alone or TGF- (5 ng/ml) with one of the following: DMSO (0.1%), PPAR agonist WY-14643 (20 µM), 15d-PGJ2 (10 µM), and rosiglitazone (20 µM). Cells were treated on day 0 and 2 and harvested on day 5. Type I collagen content was measured by commercial ELISA (Chondrex, Redmond, WA).
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay for cell viability.
The effect of the PPAR agonists on cell viability was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (27). Fibroblasts were plated in triplicate at a density of 5,000 cells per well and treated with TGF- and PPAR agonists for 72 h at the same concentrations previously used. Production of the colored reaction product was measured at 560 nm, and the results were normalized to the negative control.
Dominant-negative PPAR adenovirus infection.
A replication-deficient adenovirus containing a mutated (L466A) mouse dominant-negative PPAR1 gene was a kind gift from Drs. E. J. Lee and J. L. Jameson (Northwestern University) (28). The dominant-negative adenovirus and a control adenovirus with no inserted transgene (AdDL70) (31) were used to infect primary human pulmonary fibroblasts at a multiplicity of infection (MOI) of 30 plaque forming units (pfu) per cell. After 24 h, TGF- and PPAR agonists 15d-PGJ2 (5 µM) and rosiglitazone (20 µM) were added to the media, and the cells were incubated at 37°C for 72 h. Differentiation to myofibroblasts was assessed by -SMA ICC and Western blot analysis described above.
PPAR luciferase reporter assay.
Primary lung fibroblasts cultured in 12-well plates were cotransfected with a PPAR-luciferase reporter construct containing three PPREs (a gift from Dr. Brian Seed, Harvard University) (19) and a -galactosidase control construct (a gift from Dr. T. Gasiewicz, University of Rochester) (35) using Fugene 6 (Roche Applied Science, Indianapolis, IN). After 24 h, the cells were treated with 15d-PGJ2 (5 µM) or rosiglitazone (20 µM) in serum-reduced medium (2.5% FBS) and harvested after a further 24-h incubation. Luciferase activity was measured using a luciferase assay system (Promega, Madison, WI) in a luminometer (Packard Instruments, Meriden, CT) and normalized to -galactosidase activity (Promega). The experiments were carried out on quadruplicate wells.
Statistics. All data are expressed as means ± SE. A Student's unpaired t-test and ANOVA were used to establish statistical significance. Results were considered significant if P < 0.05.
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RESULTS |
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and RXR protein are expressed by primary human lung fibroblasts.
To determine whether primary human fibroblasts express PPAR and RXR protein and therefore have the potential to respond to PPAR agonists, primary human lung fibroblasts from five different patients were analyzed by Western blot for PPAR and RXR protein expression. As shown in Fig. 1A, all fibroblast lines robustly expressed both PPAR and RXR at the expected mass of 64 and 65 kDa, respectively.
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/RXR transcriptional activation system was active in lung fibroblasts, primary cultures were transfected with a PPAR-dependent luciferase reporter construct and treated with 15d-PGJ2 or rosiglitazone. Both PPAR agonists activated the reporter, demonstrating that primary human lung fibroblasts are competent to carry out PPAR-dependent transcriptional activation (Fig. 1B).
PPAR agonists suppress TGF- stimulated myofibroblast differentiation.
To determine whether PPAR agonists block TGF--induced differentiation of lung fibroblasts to myofibroblasts in vitro, we selected two different primary human lung fibroblast strains and treated them with TGF- and the PPAR agonists 15d-PGJ2, ciglitazone, or rosiglitazone at different concentrations. Representative data from one fibroblast strain are shown. Myofibroblast differentiation was assessed by ICC and Western blot analysis for -SMA, a marker of myofibroblast differentiation. Cultured fibroblasts treated with media alone had a few constitutively positive -SMA cells (Fig. 2A), whereas fibroblasts treated with TGF- demonstrated significant differentiation to myofibroblasts with an increase in -SMA expression (Fig. 2B). All three PPAR agonists inhibited myofibroblast differentiation in both fibroblast strains tested. 15d-PGJ2 and rosiglitazone were the most effective, as seen by the decrease in the number of -SMA positive cells (Fig. 2, E–H). Treatment with a PPAR agonist (WY-14643) did not inhibit TGF--driven myofibroblast differentiation (Fig. 2D).
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-SMA protein from parallel fibroblast cultures. Cells treated with TGF- expressed the highest levels of -SMA protein (Fig. 3A). The most potent -SMA suppression was observed with 15d-PGJ2 treatment. The synthetic PPAR agonist rosiglitazone also significantly decreased expression of -SMA protein. Again, the PPAR agonist did not reduce -SMA protein expression (Fig. 3A). Densitometry analysis normalized to GAPDH indicates lung fibroblasts treated with TGF- exhibited a 25-fold increase in -SMA expression over untreated control fibroblasts, an indication of myofibroblast differentiation (Fig. 3B). The PPAR agonist 15d-PGJ2 inhibited >95% of the TGF- stimulated -SMA induction, whereas rosiglitazone inhibited 40% of the TGF- stimulated -SMA induction (Fig. 3B).
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agonists inhibit production of type I collagen in human lung fibroblasts treated with TGF-.
Collagen is one of the major extracellular matrix component in fibrotic tissues (29). To determine whether PPAR agonists inhibit pulmonary fibroblast production of collagen, cells were treated with TGF- and PPAR agonists and collagen protein was assayed by ELISA. Untreated normal primary human pulmonary fibroblasts synthesize little collagen. However, treatment with TGF- significantly increased type I collagen production in human lung fibroblasts (Fig. 4). The PPAR agonist WY-14643 had no effect on the production of type I collagen. In contrast, 15d-PGJ2 and rosiglitazone significantly inhibited TGF--induced synthesis of type I collagen by lung fibroblasts compared with the TGF--treated cells to P < 0.01 (Fig. 4).
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agonists.
One simple explanation for the reduction in -SMA and collagen production is that PPAR agonists are toxic. To rule out this possibility, viability of human lung fibroblasts treated with PPAR agonists was measured by MTT assay. Viable cells actively cleave the MTT reagent and form a colored precipitate the appearance of which is proportionate to the number of viable cells (27). Results are shown as a percentage of the negative control, cells with only media. As Fig. 5 demonstrates, there was no difference in cell viability among treatment conditions. In particular, there was no evidence of cell toxicity in fibroblasts exposed to PPAR agonists. To further demonstrate that the agonists do not affect cell viability, fibroblasts were treated with the PPAR agonists for 3 days, after which the medium was changed and TGF- was added for a further 3 days. TGF- stimulated myofibroblast differentiation with equal efficiency in cells previously treated with 15d-PGJ2, rosiglitazone, or ciglitazone as control cells, demonstrating that at the doses used the PPAR agonists are nontoxic and the inhibition of differentiation is reversible (data not shown).
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protein partially reverses PPAR agonist-induced inhibition of myofibroblast differentiation.
Some anti-inflammatory effects of PPAR agonists may be independent of PPAR activation (6, 10). To determine whether the effects of PPAR agonists on myofibroblast differentiation are dependent or independent of PPAR activation we used a dominant-negative approach. Primary human lung fibroblasts were transfected with a replication-deficient adenovirus expressing a mutated dominant-negative PPAR gene that binds to the ligand but does not activate transcription at the PPRE (28). An adenovirus with no inserted transgene was used as a control. At an MOI of 30 pfu/cell, >90% of fibroblasts were infected (data not shown). The fibroblasts were then treated with TGF- and PPAR agonists to determine whether the dominant-negative PPAR would prevent the inhibition of TGF--driven myofibroblast differentiation by PPAR agonists. Myofibroblast differentiation was determined by ICC and Western blot analysis. Fibroblasts infected with the control virus and treated with TGF- differentiated to myofibroblasts as expected (Fig. 6A), and differentiation was inhibited by 15d-PGJ2 (Fig. 6B) or rosiglitazone (Fig. 6C), as previously shown. Fibroblasts infected with the dominant-negative PPAR adenovirus also differentiate to myofibroblasts when treated with TGF- (Fig. 6D). However, the dominant-negative PPAR blocks the ability of 15d-PGJ2 or rosiglitazone to inhibit myofibroblast differentiation (Fig. 6, E and F). This was confirmed by Western blot analysis (Fig. 6, G and H). Fibroblasts treated with TGF- and 15d-PGJ2 expressed 11% of the amount of -SMA expressed by cells treated with TGF- alone. The dominant-negative virus increased -SMA expression to 37% of the positive control, blocking 30% of the inhibitory effect of 15d-PGJ2. Although rosiglitazone is less effective at inhibiting -SMA expression (52% of the positive control), the dominant-negative virus increased -SMA levels to 88% of control, blocking 75% of the effects of rosiglitazone.
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agonists is largely via a PPAR-independent mechanism.
To further investigate the PPAR dependence of the antifibrotic effects of PPAR agonists, fibroblasts were treated with TGF-, 15d-PGJ2, and GW-9662, a highly specific and irreversible PPAR antagonist (15). GW-9662 completely inhibits 15d-PGJ2-driven differentiation of human fibroblasts to adipocytes (C. W. O'Laughlin and R. P. Phipps, personal communication), a PPAR-dependent process, as well as PPAR-dependent gene transcription in human chondrosarcoma cells (26). However, GW-9662 had no effect on the ability of 15d-PGJ2 to inhibit TGF- driven myofibroblast differentiation and -SMA expression (Fig. 7).
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DISCUSSION |
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(12, 33). Furthermore, we and others have shown that TGF- is a potent stimulus for myofibroblast differentiation and induction of pulmonary fibrosis in vivo (22, 23, 31). This study clearly demonstrates that PPAR agonists inhibit TGF- stimulated myofibroblast development and collagen production in primary human pulmonary fibroblasts, highlighting PPAR agonists as exciting potential antifibrotic therapies.
Data are presented illustrating for the first time that primary human pulmonary fibroblasts express abundant PPAR and RXR proteins and are capable of PPAR-dependent transcriptional regulation (Fig. 1). When exposed to both natural and synthetic PPAR agonists, fibroblasts exposed to TGF- are significantly inhibited from transdifferentiating to the myofibroblast phenotype. ICC and Western blot analysis for the myofibroblast marker -SMA demonstrate that normal primary human lung fibroblast strains contain few myofibroblasts (Fig. 2A). However, upon exposure to TGF-, myofibroblast differentiation is promoted and cells express -SMA (Fig. 2B). With the addition of the PPAR agonists 15d-PGJ2, ciglitazone, or rosiglitazone, -SMA expression is potently inhibited (Figs. 2, E–H, and 3). Inhibition of myofibroblast differentiation is specific to PPAR agonists, as treatment with a PPAR agonist, WY-14643, or vehicle controls does not attenuate -SMA expression and myofibroblast development (Fig. 2, C and D).
To exclude the possibility that the reduction in the level of -SMA protein in cells treated with TGF- and PPAR agonists is due to cell death, an MTT assay was performed. PPAR agonists had no effect on cell viability (Fig. 5), thus excluding cytotoxicity as a cause for reduction in -SMA expression and myofibroblast development.
Of the PPAR agonists tested, 15d-PGJ2 was the most potent inhibitor of -SMA expression and myofibroblast development. This may be a reflection of the fact that 15d-PGJ2 has both PPAR-dependent and -independent mechanisms of action. For example, the effects of 15d-PGJ2 on monocytes and lymphocytes may be mediated by PPAR-independent mechanisms, such as inhibition of transcription factors like NF-B (10, 32). In contrast, synthetic PPAR agonists such as rosiglitazone have activities that are more specific to the PPAR receptor. Therefore, the extent to which the antifibrotic effects of 15d-PGJ2 are dependent or independent on PPAR may determine the relative effectiveness of different agonists.
To address whether PPAR agonist-mediated inhibition of myofibroblast differentiation is mediated through a PPAR-dependent mechanism, dominant-negative genetic and pharmaceutical approaches were used to block the activity of endogenous PPAR. A replication-deficient adenovirus containing a dominant-negative PPAR gene was used to infect pulmonary fibroblasts and overexpress a mutated dominant-negative protein that binds to PPRE on DNA but does not initiate transcription. Under conditions in which >90% of infected fibroblasts express the dominant-negative PPAR, the dominant-negative PPAR only partially inhibits the antifibrotic effects of 15d-PGJ2 and rosiglitazone (Fig. 6). As determined by ICC for -SMA, the dominant-negative PPAR adenovector partially reverses the effect of 15d-PGJ2 (Fig. 6E) and almost completely reversed the effect of rosiglitazone (Fig. 6F). These results were confirmed by Western blot, as the dominant-negative PPAR reversed 75% of the -SMA inhibition by rosiglitazone and 30% of the -SMA inhibition of 15d-PGJ2. GW-9662, an irreversible PPAR antagonist, had no effect on suppression of -SMA expression by 15d-PGJ2 at a concentration that completely inhibits PPAR-dependent differentiation of fibroblasts to adipocytes (Fig. 7). Together, these results argue that the ability of 15d-PGJ2 and rosiglitazone to inhibit myofibroblast differentiation is mediated by both PPAR-dependent and -independent mechanisms but that PPAR-independent mechanisms likely predominate. These pathways are currently under investigation.
Excessive collagen accumulation is another key feature of fibrosis, and the pulmonary myofibroblast is one of the most important producers of excess collagen in pulmonary fibrosis (29). To determine if collagen production could be attenuated by PPAR agonists we treated primary lung fibroblasts with TGF- and either 15d-PGJ2 or rosiglitazone and examined production of type I collagen in vitro (Fig. 4). Normally, pulmonary fibroblasts produce low levels of type I collagen. With TGF- exposure there is an increase in collagen production by differentiated myofibroblasts. Treatment of lung fibroblasts with either 15d-PGJ2 or rosiglitazone potently and significantly reduced TGF--stimulated collagen production. This is a crucial finding, as excess collagen production is one of the most important pathological abnormalities of fibrotic lungs and is likely responsible for many of the physiological abnormalities, including restrictive pulmonary function abnormalities. Interestingly, although 15d-PGJ2 was a more potent inhibitor of -SMA expression, rosiglitazone was demonstrated to be a more potent inhibitor of collagen synthesis than 15d-PGJ2. This suggests that different profibrotic activities of fibroblasts (myofibroblast differentiation and collagen deposition) are regulated differently by PPAR agonists. It should be noted that rosiglitazone is a more specific PPAR agonist than 15d-PGJ2 (8, 25) and that the dominant-negative PPAR is more effective in reversing the effects on -SMA expression of rosiglitazone than 15d-PGJ2. Thus it is possible that while the effects of the PPAR agonists on myofibroblast differentiation and -SMA expression are largely mediated by a PPAR-independent mechanism, the agonists affect collagen synthesis by a largely PPAR-dependent mechanism.
The mechanisms by which PPAR agonists inhibit myofibroblast differentiation are under further investigation. One simple possibility is that the agonists directly interfere with TGF- signaling, preventing the fibroblasts from receiving profibrotic signals. TGF- signaling is mediated by phosphorylation of Smad2 and Smad3 by the TGF- receptor (11). However, Smad2/3 phosphorylation is not altered in TGF--treated lung fibroblasts exposed to 15d-PGJ2 or rosiglitazone (data not shown). Thus inhibition of myofibroblast differentiation may occur downstream of the initial TGF- signaling events or involve non-Smad-mediated TGF- signaling. One possible mechanism for the interaction between the TGF- and PPAR pathways has recently been reported (14, 16, 18, 26). In glomerular mesangial cells the PPAR agonist pioglitazone inhibits TGF--induced AP-1 binding activity, thus reducing TGF--stimulated increases in fibronectin mRNA. Understanding similar potential interactions of PPAR and TGF- in pulmonary fibroblasts will increase our understanding of the mechanisms by which PPAR agonists have antifibrotic activities.
Overall, our results convincingly demonstrate that PPAR agonists potently interrupt two of the most important profibrotic effects of TGF- on normal human primary pulmonary fibroblasts, the induction of myofibroblasts, and stimulation of excess collagen production. These data are exciting and suggest that the PPAR pathway is likely a very important future target for therapy of fibrosis of the lungs and other tissues. An added advantage of using PPAR agonists as novel antifibrotics is that these drugs are currently available for use in patients with diabetes. This will potentially facilitate their rapid translation to use in patients with lung fibrosis, for whom few effective therapies currently exist.
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ACKNOWLEDGMENTS |
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1. The authors also thank Dr. T. Gasiewicz (University of Rochester) for the -galactosidase expression plasmid and Dr. Brian Seed (Harvard University) for the PPRE-luciferase plasmid. L. E. Daugherty is currently at Sunrise Children's Hospital, Las Vegas, NV.
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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.
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0.03).
