Abstract Increasing evidence suggests that cytokines such as interleukin-1β (IL-1), IL-4, and IL-8 may play an important role in the chronic inflammation and cellular growth observed in cardiovascular diseases. The lipoxygenase (LO) pathway of arachidonate metabolism has also been related to the pathology of hypertension and atherosclerosis. LO products have chemotactic, hypertrophic, and mitogenic effects in vascular cells, and the LO enzyme has been implicated in the oxidation of LDL. Furthermore, earlier studies have shown that vascular smooth muscle cell (VSMC) growth factors such as angiotensin II and platelet-derived growth factor can increase LO activity and expression in VSMCs. In the present study, we have examined whether vasoactive and inflammatory cytokines such as IL-1, IL-4, and IL-8 can modulate 12-LO activity and expression in porcine VSMCs and also whether they have growth-promoting effects in these cells. Treatment of porcine VSMCs with these cytokines led to significant increases in the levels of a cell-associated 12-LO product, 12-hydroxyeicosatetraenoic acid, as well as intracellular 12-LO enzyme activity. Furthermore, each of these cytokines led to a dose-dependent increase in 12-LO mRNA expression (333-base pair PCR product) as well as 12-LO protein expression (72 kD). In addition, all three interleukins could induce significant increases in VSMC DNA synthesis as well as proliferation. These results suggest that these cytokines have mitogenic effects in VSMCs and are also potent positive regulators of the 12-LO pathway. Thus, enhanced 12-LO activity and expression may be a key mechanism for cytokine-induced VSMC migration and proliferation.
The pathogenesis of atherosclerosis and hypertension involves several key events, including abnormal VSMC proliferation and migration, mediated by a complex interaction of numerous growth factors, cytokines, lipoproteins, and lipids.1 2 Recent studies have indicated that VSMC growth and migration can be affected not only by factors released by platelets but also by cytokines released by monocytes, macrophages, and T cells.3 4 Chronic inflammatory cells, including T cells and macrophages, are present at all stages of atherogenesis,5 and the cytokines released by them may in turn stimulate the release of more cytokines and growth factors by the same or adjoining cells and may therefore also play a key role in vascular disease and atherosclerosis.6
The interaction of cytokines with their cell surface receptors leads to a multitude of signaling events including the activation of several phospholipases, which in turn can lead to the release of arachidonic acid. Arachidonic acid has been suggested to be a potential mitogenic signal, and it is also the precursor for several eicosanoids with potent biological effects including cellular inflammation and growth.7 Arachidonic acid can be metabolized by three major pathways: the cyclooxygenase pathway, which leads to the formation of prostaglandins and thromboxane; the LO pathway, which forms hydroxyeicosatetraenoic acids (HETEs) and leukotrienes; and the cytochrome P-450 monoxygenase pathway, which leads to the formation of epoxides.8
Arachidonate 12-LO introduces molecular oxygen into the 12-position of arachidonic acid to yield 12-hydroperoxyeicosatetraenoic acid, which is an unstable intermediate and is rapidly reduced to 12-HETE. Studies show that there are at least two major isoforms of 12-LO, a platelet type and a leukocyte type.9 10 11 12 The leukocyte-type 12-LO has been detected in porcine leukocytes,13 VSMCs,14 human adrenal glomerulosa cells,15 human monocytes, and endothelial and VSMCs.16
Increasing evidence has implicated the LO enzymes and their products such as HETEs in the pathogenesis of hypertension and atherosclerosis. The LO enzyme has been suggested to mediate the oxidative modification of LDL17 and has been shown to be expressed in macrophage-rich areas of atherosclerotic lesions.18 Analysis of the lipid oxidation products in human atherosclerotic lesions revealed that the oxidation of polyunsaturated fatty acids therein was mainly mediated by the LO enzyme.19 The LO pathway has also been implicated in the vasopressor and renin inhibitory effects of angiotensin II.20 21 Furthermore, inhibition of the LO pathway could reduce blood pressure in renovascular hypertensive rats22 and in the SHR rats, which also had increased 12-LO activity compared to the nonhypertensive control rats.23 LO products have potent chemotactic effects in VSMCs, inducing VSMC migration at concentrations as low as 1 pmol/L.24
We have recently shown that angiotensin II, a potent VSMC growth factor, can increase 12-HETE formation and intracellular 12-LO activity, as well as the expression of the leukocyte-type 12-LO mRNA and protein in VSMCs.14 16 Furthermore, the hypertrophic effects of angiotensin II were attenuated by a specific LO blocker, and the 12-LO product 12-HETE had direct hypertrophic effects in VSMCs.25 We have also recently shown that platelet-derived growth factor, a potent VSMC mitogen and chemoattractant, caused marked increases in 12-LO activity and expression in VSMCs26 and that the LO pathway plays a role in the chemotactic effects of platelet-derived growth factor in VSMCs.26 In view of the cardiovascular effects of cytokines, we have now examined whether cytokines such as interleukin-1β (IL-1), IL-4, and IL-8 have the ability to induce the 12-LO enzyme in porcine VSMCs.
Cytokines can affect several enzymes and factors that regulate vascular tone and VSMC proliferation and migration. IL-1 has mitogenic effects in VSMCs,3 and this property has also been attributed to an increase in PDGF-A chain production.27 IL-1 can induce the production of IL-1 itself as well as IL-6 in VSMCs, thus setting up an amplification loop.3 28
IL-4, mainly a product of activated T lymphocytes,5 has several properties relevant to the pathogenesis of atherosclerosis. It can induce the expression of vascular cell adhesion molecule 1 in cultured endothelial as well as VSMCs.29 30
IL-8, a product of monocytes, macrophages, and other cells,31 can induce VSMC mitogenesis as well as migration.32 Furthermore, IL-8 protein, mRNA, and bioactivity were shown to be upregulated in atherosclerotic plaques.33
The present studies show that some of the vascular effects of these cytokines may be mediated by upregulation of the 12-LO pathway.
Recombinant human IL-1β, IL-4, and IL-8 were obtained from R & D Systems. The LO products and inhibitors were from BIOMOL Research Laboratories. All reagents for cell culture were from Irvine Scientific.
Culture of Porcine Aortic Smooth Muscle Cells
Primary cultures of PVSMC were obtained as described earlier14 and used up to passage 6. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing normal glucose concentrations (5.5 mmol/L) and 10% FCS.
Measurement of the LO Product 12-HETE by Radioimmunoassay
Confluent cells in 100-mm dishes were serum depleted by placing in DMEM/HEPES containing 0.2% BSA and 0.4% FCS for 24 hours. Just prior to the experiment, the cells were placed in media containing 0.2% BSA only, preincubated for 20 minutes at 37°C, treated with the cytokines, and then incubated for various time periods. Washed cell pellets were first deacylated to obtain cell-associated esterified HETEs as described earlier.14 HETEs released into the medium and cell-associated HETEs in the deacylated cell pellets were extracted as described.14 12-HETE in the supernatant medium and cell extracts were quantitated by a specific radioimmunoassay.14 The 12-HETE antiserum (Perseptive Diagnostics) is specific for 12(S)-HETE, with less than 0.1% cross-reactivity with 12(R)-HETE, 0.3% with 15-HETE, 0.2% with 5-HETE, <0.1% with thromboxane B2, and 0.1% with arachidonic acid. Nonspecific binding is <6%, with an assay blank of 7 pg/mL. The sensitivity of the assay is 10 pg/mL with an intra- and interassay variation of 7% and 10%, respectively.
Measurement of Intracellular LO Activity
PVSMCs in 100-mm dishes were serum-depleted for 24 hours and then treated with the cytokines for 6 hours. 12-LO activity in cell sonicates was then assayed by examining the conversion of substrate arachidonic acid to 12-HETE as described earlier.14 26 Briefly, the sonicates were incubated with sodium arachidonate for 8 minutes, extracted,14 26 and then subjected to reverse-phase HPLC as described.26 Quantification of 12-HETE peak heights was by a Shimadzu C-R5A integrator (Shimadzu Instrument Co). The recovery of added [3H]12-HETE as an internal standard taken through the entire process of extraction, HPLC, and fraction collection was 72±3%.
Incubations for 12-LO Protein or mRNA Expression
Nearly confluent PVSMCs in 100-mm dishes were serum depleted for 24 hours by placing in DMEM/HEPES containing 0.2% BSA+0.4% FCS. This medium was then freshly added alone or with the respective interleukins. At the end of the incubation period, the cells were processed for immunoblotting or RNA extraction as described below.
Electrophoresis and Western Immunoblotting
Cell pellets were lysed, and the cytosolic protein (30- to 50-μg aliquots) was subjected to electrophoresis and immunoblotting as described earlier.14 15 Protein blots were incubated with a polyclonal antibody (1:400) raised to a specific 12-LO peptide with amino acid sequence 646-662 of porcine leukocyte 12-LO.13 26 Blots were then washed and incubated with an alkaline phosphatase–labeled second antibody (1:30 000). Detection was by the Western Light Chemiluminescent system (Tropix Inc). Blots were quantitated with a computerized densitometer (SCISCAN 5000, United States Biochemical), and values were expressed as arbitrary absorbance units.
12-LO cDNA, Oligonucleotide Primers, and Probes for PCR
pUC19 plasmid with the cDNA for porcine leukocyte 12-LO was originally a generous gift from Dr T. Yoshimoto, Japan.13 15 All of the oligonucleotides, including human GAPDH, were synthesized on an Applied Biosystems DNA synthesizer at the Beckmann Research Center nucleotide synthesis facility and were purified by polyacrylamide gel electrophoresis. The oligonucleotide sequences are as shown earlier14 15 and were based on known gene sequences and selected from regions displaying the most divergence between porcine 12- and human 15-LO sequences due to their high homology.
Amplification of Reverse-Transcribed RNA Using RT-PCR
PVSMCs that had been treated with or without cytokines were subjected to total RNA extraction followed by RT-PCR to specifically amplify porcine leukocyte-type 12-LO as described earlier.14 26 GAPDH mRNA amplification was used as an internal control for RNA quantity and amplification efficiency. Blank reactions with no RNA template or with no reverse transcriptase were carried out through the RT and PCR steps. Porcine leukocyte 12-LO cDNA amplification was used as positive control. The PCR products were then subjected to Southern blotting followed by hybridization to detect the leukocyte-type 12-LO or GAPDH as described.14 15 The ratio of 12-LO mRNA expression (333-bp PCR product) to the corresponding GAPDH expression (284-bp PCR product) was calculated in each sample. Washing and hybridization conditions were developed to distinguish between the PCR products of human 15-LO from those of porcine leukocyte 12-LO.15
Cellular Proliferation Studies
For [3H]thymidine incorporation studies to evaluate DNA synthesis, PVSMCs in 24-well dishes were made quiescent by placing them in medium containing 0.4% FCS and 0.2% BSA for 24 hours. This medium was then freshly replaced, and the cells were preincubated with 2.5 μmol/L indomethacin (cyclooxygenase blocker). The cytokines were then added, and cells were incubated for 36 hours with [3H]thymidine (1 μCi/mL) added during the last 18 hours. In some experiments, the cells were also preincubated for 15 minutes with the LO inhibitor baicalein or the corresponding vehicle (0.1% DMSO) before cytokine addition. At the end of the incubation period, the cells were washed with PBS, followed by cold 10% trichloroacetic acid two times. Washed cells were then solubilized with 0.5 mL 0.3N NaOH. Radioactivity in the resulting solutions was quantitated by scintillation counting.
For experiments examining effects of the cytokines on cell proliferation, PVSMCs were plated in 6-well dishes (15 000 cells per well) for 48 hours in DMEM+10% FCS. The cells were then placed in fresh medium containing 0.2% BSA and 0.4% FCS for 24 hours. This medium was then freshly replaced along with the cytokines with or without indomethacin (2.5 μmol/L). Medium along with fresh additions were replaced every 48 hours. After 6 days, cells were trypsinized, and cell numbers were quantified on a Coulter counter (Coulter Corp).
Western immunoblots and autoradiograms of Southern blots were analyzed with a computerized densitometer (SCISCAN 5000, United States Biochemical). The values are obtained as arbitrary absorbance units. The other results are expressed as mean±SEM from combined experiments as noted in the corresponding legend. Student’s t tests and ANOVA with Dunnett or Tukey-Kramer multiple comparison tests were used to analyze the data using the INSTAT software (Graphpad Software Inc).
Effect of IL-1, IL-4, and IL-8 on 12-HETE Levels in PVSMCs
We initially examined whether treatment of the PVSMC with the interleukins can affect the levels of released or cell-associated immunoreactive 12-HETE. Fig 1⇓ shows that a 6-hour treatment with IL-1β, IL-4, or IL-8 elicited a significant increase in cell-associated 12-HETE levels with each one of the cytokines. Initial time course studies revealed an increase in cell-associated 12-HETE levels by 2 hours, which peaked by 6 hours and then gradually declined. The increases in cell-associated 12-HETE levels at 6 hours were fully blocked by 10-minute pretreatment of the cells with a 12-LO inhibitor, baicalein 10−5 mol/L (basal, 861±55 pg/106 cells; IL-1, 1132±60 pg; IL-4, 1594±18 pg; IL-8, 1409±90 pg; IL-1+baicalein, 780±36 pg; IL-4+baicalein, 916±81 pg; IL-8+baicalein, 901±44 pg; all P<.01 vs respective IL). Baicalein alone did not alter basal 12-HETE (904±61 pg/106 cells). There was no significant increase in the levels of released 12-HETE into the medium at these time periods with any of the cytokines.
Effect of IL-1β, IL-4, and IL-8 on 12-LO Enzyme Activity in PVSMCs
We next examined whether these interleukins can alter intracellular 12-LO enzyme activity in the VSMCs, ie, the conversion of exogenously added cold arachidonate to 12-HETE by crude enzyme in cell sonicates. Fig 2a⇓ shows the HPLC tracings of extracts of sonicates of cells that had been treated with or without IL-1 for 24 hours. The first panel depicts the retention times of the authentic cold standards, 12-HETE and 15-HETE. The second panel shows the HPLC tracing of 12-LO activity in control cells, where a distinct peak with the same retention time as 12-HETE is seen. Furthermore, in the next panel, it is seen that treatment of the cells with IL-1 caused an increase in 12-LO activity of 1.6-fold over control. An enzyme blank seen in the last panel on the right was run as a control for nonspecific, nonenzymatic oxidation of the substrate arachidonic acid. Under these conditions, there does not seem to be any increase in the 15-HETE peak. The identity of the 12-HETE peak in the HPLC tracings in this assay was confirmed by (1) comigration with authentic cold standard; (2) observing a quantitative increase in the 12-HETE peak height when a known amount of authentic cold 12-HETE (5 ng) was injected along with a sample; and (3) by radioimmunoassay of the 12-HETE fraction obtained after HPLC separation.
Fig 2b⇑ and 2c⇑ show the representative results obtained with cells treated with IL-4 and IL-8, respectively. Similar to IL-1, both these cytokines also caused increases in 12-LO activity (1.5- and 1.3-fold over control, respectively).
The Table⇓ shows cumulative results of the effects of these three interleukins on 12-LO enzyme activity from several experiments. 12-LO activity is represented as height of the 12-HETE peak in reverse-phase HPLC as fold over control. All three cytokines induced significant increases in the 12-HETE peak height when used at 5 ng/mL each.
Effect of IL-1β, IL-4, and IL-8 on 12-LO mRNA Expression
To determine whether these interleukins can regulate the expression of 12-LO mRNA in PVSMCs, we examined 12-LO mRNA levels using a specific RT-PCR method in cells that had been treated for 20 hours with the cytokines. We have shown earlier that PVSMCs express a leukocyte-type 12-LO but not 15-LO.14 Fig 3a⇓ shows the dose-response effect of IL-1β on leukocyte-type 12-LO mRNA expression in PVSMCs. The size of the amplified product is 333 bp. The representative Southern blot shown in Fig 3a⇓ shows that basal expression of the 12-LO mRNA 333-bp PCR product is very low. However, treatment with IL-1 at concentrations from 1 to 5 ng/mL led to a marked dose-dependent increase in 12-LO mRNA expression. The positive control for PCR, 12-LO cDNA amplification, is seen at the far right, whereas a negative control without RNA is seen in the previous lane. The results were normalized to the intensity of the band of the internal control for PCR, ie, GAPDH mRNA (284-bp PCR product, lower panel of Fig 3a⇓).
We similarly conducted experiments to evaluate the effects of IL-4 and IL-8 on 12-LO mRNA expression. The representative results, seen in the Southern blots in Figs 3b⇑ and 3c⇑, respectively, indicate that, similar to IL-1, IL-4 and IL-8 also evoke potent dose-dependent increases in 12-LO mRNA expression.
The Table⇑ shows combined data from several experiments depicting the effect of these cytokines on 12-LO mRNA after normalizing for GAPDH mRNA expression. It shows that the three cytokines can lead to increases of 7- to 10-fold over control in 12-LO mRNA expression in the PVSMCs.
Effect of IL-1β, IL-4, and IL-8 on 12-LO Protein Expression
To determine whether the cytokine-induced increases of 12-LO mRNA expression are accompanied by corresponding increases in 12-LO protein expression, we evaluated the effects of IL-1, IL-4, and IL-8 (24-hour treatment) on leukocyte-type 12-LO expression, which we have shown previously to be present in PVSMCs.14 Fig 4a⇓ shows that IL-1 leads to a dose-dependent increase in 12-LO protein expression (72 kD). Figs 4b⇓ and 4c⇓ depict the dose-dependent effects of IL-4 and IL-8 on 12-LO protein expression. It is evident that these two ILs also evoke marked increases in 12-LO protein expression at doses ranging from 2.5 to 10 ng/mL. The results suggest that these cytokines can regulate leukocyte-type 12-LO expression at the transcriptional as well as translational levels.
The Table⇑ shows a densitometric representation of the effect of the three cytokines on 12-LO protein expression obtained from multiple experiments and reveals a significant increase of 1.7- to 2.5-fold over control.
Growth-Promoting Effects of IL-1, IL-4, and IL-8 in PVSMCs
Evidence suggests that IL-1 can have mitogenic effects in VSMCs, particularly in the presence of the cyclooxygenase inhibitor indomethacin, which serves to block the production of growth-inhibitory prostanoids by the cytokine. In the present study, we have examined the functional significance of interleukin action on 12-LO in VSMCs by first determining whether they have mitogenic effects and then evaluating the potential role of 12-LO in these mitogenic effects.
Fig 5⇓ shows the effects of the three cytokines on PVSMC DNA synthesis as evaluated by [3H]thymidine incorporation. The experiments were performed in the presence of 2.5 μmol/L indomethacin. The first, third, and fifth bars indicate that the three cytokines IL-1, IL-4, and IL-8, respectively, lead to significant increases in thymidine incorporation, with IL-4 appearing to be the most potent of the three. In fact, this is the first demonstration of the mitogenic effect of IL-4 in VSMCs. To determine the potential role of the 12-LO pathway in these growth-promoting effects, we examined the effects of a specific LO inhibitor, baicalein at 10 μmol/L, on cytokine-induced DNA synthesis in the presence of indomethacin. The results of these experiments are also seen in Fig 5⇓, depicted by the second, fourth, and sixth bars. Thus, in each case, pretreatment with baicalein led to a significant attenuation of the cytokine-induced effects. The blockade was complete in the case of IL-1 (black shaded bars) and partial in the case of IL-4 (hatched bars) and IL-8 (square bars). Baicalein alone at this concentration did not significantly affect basal thymidine incorporation (bar on the extreme right). Baicalein at this dose could also block cytokine-induced 12-HETE formation, similar to our earlier observations.14 26
It is, however, important to determine whether these increases in DNA synthesis represent true mitogenic events. We therefore evaluated changes in cell numbers after chronic exposure of the cells to these cytokines for 6 days in medium containing very low amounts of serum. Fig 6⇓ shows the results obtained both in the absence (empty bars) and the presence (bold bars) of indomethacin. It is seen that all three cytokines lead to significant increases in VSMC proliferation (open bars). Fig 6⇓ also shows that in these experiments where the VSMCs were chronically exposed to interleukins for several days, the addition of indomethacin did not further enhance the mitogenic effects (closed bars). A similar observation was also made by Libby et al3 in earlier studies with IL-1β.
Although there is no doubt that the etiology of atherosclerosis is multifactorial, several lines of evidence support the role of inflammatory cytokines such as IL-1β, IL-4, and IL-8 because of their key roles in inflammation and immune reactions. Cytokines have growth-promoting and chemotactic effects in VSMCs and also increase the expression of leukocyte adhesion molecules on endothelial cells and VSMCs. High levels of cytokines have also been observed in atherosclerosis. The signaling mechanisms of these cytokines can thus provide clues to potential mechanisms for the progression of the atherosclerotic plaque.
Very few studies have examined the effects of cytokines on the LO pathway. It was recently shown that treatment of monocytes with IL-4 or IL-13 could activate the 15-LO pathway by inducing 15-LO mRNA as well as protein expression.34 35 The LO pathway has been implicated in the oxidation of LDL.17 18 We have recently shown that VSMC growth factors such as platelet-derived growth factor and angiotensin II are potent inducers of 12-LO activity and expression in porcine and human VSMCs.14 16 26 In addition, the LO pathway seems to play a role in the hypertrophic effects of AII and the chemotactic effects of platelet-derived growth factor in VSMC.25 26 The results indicate that LO in vascular and mononuclear cells can be induced by growth factors and cytokines and may contribute to the growth of VSMCs.
In the present study, we have shown that the three cytokines tested, ie, IL-1, IL-4, and IL-8, can also activate the 12-LO pathway in porcine VSMCs. Treatment of PVSMCs with each of these cytokines led to a significant increase in cell-associated 12-HETE levels as well as an increase in intracellular 12-LO enzyme activity. In addition, these three cytokines can induce the mRNA and protein expression of this 12-LO. These results further support the potential role of the LO pathway in hypertensive and atherosclerotic processes, especially in view of the several cardiovascular effects of LO products. Although peak 12-LO mRNA and protein expression were observed between 20 and 24 hours, we have noted that their levels were upregulated as early as 6 hours. The increase in 12-LO activity seen at the 6-hour time period (shown in Figs 1⇑ and 2⇑) could, therefore, arise either from increased substrate (arachidonic acid) availability or from increased 12-LO expression. 12-LO activity also remains elevated up to 24 hours.
Earlier studies have had difficulty in observing direct mitogenic effects of these interleukins in vitro with VSMC cultures, and this has been attributed to the potent stimulatory effects of these cytokines on the cyclooxygenase pathway, which leads to the formation of growth-inhibitory prostanoids. Thus, IL-1β and IL-8 have been demonstrated to have mitogenic effects on VSMCs in the presence of the cyclooxygenase inhibitor indomethacin.3 32 The present studies have confirmed these observations in porcine VSMCs. Furthermore, we have shown for the first time that IL-4 has mitogenic effects in VSMCs and in fact was the most potent of the three. In addition, the observation that the LO inhibitor baicalein could attenuate their effects suggests that the LO pathway may, at least in part, mediate their growth effects. We have also verified that the observed increases in DNA synthesis are also accompanied by increases in cell number, thus indicating a true mitogenic response. The inflammatory effects of IL-8 have been attributed not only to its mitogenic but also to its chemotactic effects.32 Because 12-LO products such as 12-HETE are potent inducers of VSMC migration, it is also likely that they mediate the chemotactic effects of these cytokines. Thus, it is possible to envision an imbalance between activation of the LO and cyclooxygenase pathway in pathological situations, leading to an increased accumulation of inflammatory, chemotactic, and growth-promoting LO products.
The mechanisms by which LO products mediate basal or cytokine-induced VSMC growth and migration have not been evaluated in these studies, but evidence indicates that they can initiate several growth-related signaling events such as activation of oncogenes, protein kinase C, and mitogen-activated protein kinase.36 The present results suggest that LO enzymes activated by cytokines in vascular and inflammatory cells can form products that have several actions, including potent growth, chemotactic, and inflammatory effects, which therefore implicate them in the pathogenesis of diseases such as atherosclerosis, hypertension, and diabetes. We have recently developed a specific molecular technique to block 12-LO mRNA by using a specific ribozyme or catalytic RNA, which efficiently cleaves porcine leukocyte-type 12-LO.37 Hence, pharmacological as well as molecular strategies using ribozyme or antisense technology to efficiently block these pathways may serve as novel approaches to combat cardiovascular disease.
Selected Abbreviations and Acronyms
|FCS||=||fetal calf serum|
|HPLC||=||high-performance liquid chromatography|
|PCR||=||polymerase chain reaction|
|PVSMC||=||porcine vascular smooth muscle cell|
|RT-PCR||=||reverse transcription polymerase chain reaction|
|VSMC||=||vascular smooth muscle cell|
This work was supported by grants to R.N. from the National Institutes of Health (NIH), HL-48920 and PO1-HL55798, jointly funded by the NIH and the Juvenile Diabetes Foundation. J. Rosdahl was supported by an American Heart Association (California Affiliate) Student Fellowship. We thank Dr Jerry Nadler for helpful discussions and Linda Lanting for technical assistance.
- Received December 5, 1996.
- Revision received January 23, 1997.
- Accepted April 10, 1997.
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