Induction of Monocyte Chemoattractant Protein-1 in the Small Veins of the Ischemic and Reperfused Canine Myocardium
Background Healing after myocardial infarction is characterized by the presence of macrophages in the infarcted area. Since augmented monocyte influx has been implicated as a potential mechanism for improved healing after reperfusion, we wished to study the induction of monocyte chemoattractant protein-1 (MCP-1) during reperfusion.
Methods and Results The cDNA for MCP-1 was cloned from a canine jugular vein endothelial cell (CJVEC) library and exhibited 78% identity with the deduced amino acid sequence of human MCP-1. Samples of myocardium were taken from control and ischemic segments after 1 hour of ischemia and various times of reperfusion; total RNA was isolated from myocardial samples and probed with a cDNA probe for canine MCP-1. Induction of MCP-1 mRNA occurred only in previously ischemic segments within the first hour of reperfusion, peaked at 3 hours, and persisted throughout the first 2 days of reperfusion. In the absence of reperfusion, no significant MCP-1 induction was seen. Both ischemic (but not preischemic) cardiac lymph and human recombinant TNF-α induced MCP-1 in CJVECs. MCP-1 was identified by immunostaining on infiltrating cells and venular (but not arterial) endothelium by 3 hours. In contrast, in situ hybridization showed MCP-1 mRNA to be confined to the endothelium of small veins (venules) 10 to 70 μm in diameter.
Conclusions MCP-1 mRNA is induced in the endothelium of a specific class of small veins immediately after reperfusion. MCP-1 induction is confined to the previously ischemic area that has been reperfused. We suggest a significant role for MCP-1 in monocyte trafficking in the reperfused myocardium.
Early reperfusion of ischemic myocardium has become the mainstay of therapeutic intervention for patients with an evolving myocardial infarction. Conclusive evidence based on animal models shows that coronary occlusion leads to a wave front of myocardial necrosis that spreads with time of occlusion from the endocardium to the epicardial surface.1 Recent clinical trials have highlighted two important aspects of reperfusion therapy. First, early reperfusion indeed reduces infarct size, decreases ventricular dilation, and improves survival.2 Second, reperfusion even after the time period during which myocardial necrosis can be prevented (ie, no further measurable myocardial salvage) is associated with decreased ventricular dilation and enhanced survival.3
Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory reaction. The role of anti-inflammatory agents in the reduction of myocardial infarct size has been studied for almost 20 years.4 The initial studies were done with nonspecific anti-inflammatory agents that significantly reduced infarct size in a variety of animal models. However, it was soon realized that nonspecific inhibition of the inflammatory reaction also resulted in inadequate healing5 ; indeed, a clinical trial with methylprednisolone resulted in an increase in ventricular rupture.6 These observations revealed a need to better understand the cell biological factors that control postreperfusion inflammation and to dissect out the factors responsible for cellular injury from those factors critical to ventricular healing.
Healing after myocardial infarction is characterized by the presence of macrophages in the infarcted area.1 7 8 Recent animal studies have shown that reperfusion is also associated with the accelerated clearance of necrotic debris, suggesting that these macrophages might accelerate healing in this manner.9 Morita et al10 extended these observations and demonstrated that reperfusion (early or late) is associated with an increased presence of macrophages compared with the nonreperfused myocardium. Since leukocyte influx into the infarcted myocardium (without reperfusion) begins between 12 and 24 hours,7 reperfusion may therefore initiate an inflammatory response that accelerates recruitment of mononuclear monocytes that may play a crucial role in healing and remodeling.
Complement-derived chemotaxis rapidly disappears after reperfusion,11 12 so it is likely that other chemotactic mediators participate in recruitment of leukocytes into the injured myocardium. In the accompanying article,13 we provide evidence for the presence of noncomplement chemoattractants that appear within the cardiac lymph in the second and third hour of reperfusion when C5a is rapidly disappearing11 12 and suggest the potential importance of monocyte chemoattractant protein-1 (MCP-1) as a mononuclear cell chemoattractant. This report describes the induction of MCP-1 synthesis during reperfusion of the previously ischemic myocardium.
Healthy mongrel dogs (15 to 25 kg) of either sex were surgically instrumented as previously described.11 14 Anesthesia was induced with 10 mg/kg methohexital sodium IV (Brevital; Eli Lilly and Co) and maintained with the inhalational anesthetic isoflurane (Anaquest). A midline thoracotomy provided access to the heart and mediastinum, and in some experiments, cannulation of the cardiac lymph duct was then performed as previously described.14 Subsequently, a hydraulically activated occluding device and a Doppler flow probe11 15 were secured around the circumflex coronary artery just proximal or just distal to the first branch. Choice of location depended on the proximity and anatomic arrangement of lymphatic vessels, so that subsequent dissection would not damage the lymphatic system.14 In animals selected for experimental assessment of cardiac lymph, intact lymphatic vessels draining the regions of ischemic myocardium were identified by Evans blue dye (0.05 mL) injected into the free wall of the left ventricle after the occluder and flow probe were in place. The appearance of Evans blue dye in the cardiac lymph cannula confirmed the patency of the lymph vessels. Indwelling cannulas placed in the right atrium, left atrium, and femoral artery allowed blood sampling and pressure monitoring as needed. The animals were allowed to recover for 72 hours before occlusion was performed. Ischemia-reperfusion protocols were performed in awake animals as previously described.11 15 16 17 Coronary artery occlusion was achieved by inflating the coronary cuff occluder until mean flow in the coronary vessel was zero, as determined by the Doppler flow probe. After 50 minutes of occlusion, radiolabeled microspheres (for subsequent blood flow determinations) were injected into the left atrium. At the end of 1 hour, the cuff was deflated and the myocardium reperfused. Reperfusion intervals ranged from 1 to 24 hours. In some experiments, reperfusion was not instituted. Circumflex blood flow, arterial blood pressure, heart rate, and ECG (standard limb II) were recorded continuously. Analgesia was maintained with intravenously administered pentazocine (Talwin; Winthrop Pharmaceuticals). When the animals were killed, samples were taken systematically from the left ventricle, and ischemic blood flow was quantified. Control (no evidence of necrosis, normal blood flow) and ischemically injured myocardium (based on methods mentioned above and reductions in blood flow) were thus defined. The data in this article describe the findings in these two groups of segments. Segments that were initially identified as infarcted in our initial blind assessment but that had normal blood flow during occlusion did not have detectable levels of MCP-1 mRNA and therefore were analogous to control segments (data not shown).
Isolation and Culture of CJVECs: Effect of Cardiac Lymph
Cells were obtained by modification of the method of Ford et al.18 Jugular veins were everted on glass rods and incubated in collagenase solution (Boehringer Mannheim type A) for 20 minutes. Cells were collected by centrifugation and suspended in DMEM containing 5% FCS, 5% bovine calf serum, 50% μg/mL endothelial cell growth factor (Collaborative Research), 50 U/mL heparin, 1 mmol/L sodium pyruvate, and antibiotics. Cells were seeded in Primaria flasks (Becton Dickinson). After 2 to 4 days of incubation at 37°C in a CO2 incubator, areas of cells with “cobblestone” morphology were collected by scraping, transferred to gelatin-coated flasks (0.1% Difco), and grown to confluence. For incubation experiments with cardiac lymph, aliquots of lymph were obtained before, during, and after coronary occlusion. Lymph samples were collected from the cannulas in tubes containing 10 U preservative-free heparin. The samples were spun in a tabletop centrifuge at 13 000g for 5 minutes. Aliquots of supernatants were immediately frozen in liquid nitrogen and stored at −80°C until ready to use. For all incubation protocols, lymph samples, once thawed, were used immediately.
Myocardial Sampling and Calibration With Coronary Blood Flow
After the reperfusion periods, hearts were stopped by the infusion of saturated potassium chloride, removed from the chest, and sectioned from apex to base into four transverse rings ≈1 cm thick. The posterior papillary muscle and the posterior free wall were identified. Transmural myocardial samples (1.0 g) were isolated from myocardial rings and labeled as control (obtained from the anterior wall) or infarcted (obtained from the posterior papillary muscle and posterior free wall) on the basis of anatomic location within the distribution of the circumflex artery and visual inspection. Myocardial samples were then dissected into smaller pieces: first, a transmural section was taken from the middle of the sample and fixed in 10% buffered formalin for histological studies, then the top and bottom halves were cut into smaller pieces and divided into two halves. The first half was used for blood flow determinations with radiolabeled microspheres as previously described.14 19 20 21 The remaining portions of each sample were immediately frozen in liquid nitrogen. Frozen tissue samples were homogenized and processed for RNA studies. Analysis of RNA, blood flow determinations, and histopathological examinations of samples obtained from each experiment were conducted independently (in separate laboratories) and in a blinded fashion. Once the independent analyses of the data were completed, the information was gathered and a final analysis performed. The first step in this final analysis was to determine the presence or absence of a myocardial infarction in each separate experimental sample by histological examination (see below); all samples from ischemic segments were examined. A subsequent step in the final analysis of each sample was to correlate the level of MCP-1 mRNA with the blood flow quantified by microspheres.
RNA Isolation and Northern Blot Analysis
Canine myocardium of dogs subjected to the ischemia-reperfusion protocol was harvested as described above. After tissue was homogenized, total RNA was isolated by acid guanidinium thiocyanate–phenol-chloroform extraction. Subsequently, 10 to 20 μg of RNA was loaded per lane, and electrophoresis was performed in a 1% agarose/formaldehyde gel. Equal loading of lanes was assessed by photomicrographs of the ethidium bromide–stained gels. The RNA was then transferred to a charged membrane, and prehybridization and hybridization were performed with Quik-Hyb (Stratagene) according to the manufacturer's protocol using cDNA probes labeled with 32P by random hexonucleotide priming.
A partial human MCP-1 cDNA clone (ATCC) was used as a probe to screen a random-primed lipopolysaccharide-stimulated CJVEC library. Screening was performed by plaque filter hybridization methods on duplicate filters. Filters were hybridized in rapid hybridization buffer (Amersham Corp) at 64°C for 2 hours and then washed with 1×SSC/0.5% SDS at 65°C for 20 minutes followed by 0.5×SSC/0.2% SDS for 15 minutes. Hybridizing clones were plaque-purified by subsequent rounds of screening and were rescued into pBluescript SK(−) (Stratagene). Two unique clones were identified: clone 8, containing the ATG start codon, and clone 6, containing the TGA stop codon. Both clones were sequenced from double-stranded DNA templates by the dideoxynucleotide chain termination method with Sequenase version 2.0 (United States Biochemical Corp). Sequence analysis revealed an Nco I restriction endonuclease site at bp 220 that was utilized for both clones, which were then ligated to yield the full-length canine MCP-1 cDNA. Sequence analysis and database searches were done in the Molecular Biology Information Resources Center at Baylor College of Medicine.
Riboprobe Generation and Selection
Riboprobes (sense and antisense) were generated from clone 8, which contains bp 1 through 272, by use of the Genius system (Boehringer Mannheim) according to the manufacturer's protocol. Briefly, T3 and T7 RNA polymerases were used (2 U/μL) on linearized template (1 μg/20 μL) to generate the sense and antisense riboprobes, respectively. The labeled RNA was then precipitated with 3.0 mol/L sodium acetate and, after 2 washes with 75% ethanol, was resuspended in 50 μL diethylpyrocarbonate-treated water. Probe concentration was estimated by comparison of several 10-fold serial dilutions of the probe with a control labeled RNA of known concentration. After probe concentration was obtained, the probe specificity was verified by Northern blot analysis, according to the manufacturer's protocol, on membranes prepared as described previously. A colorimetric reaction was detected with the antisense probe, but none was seen with the sense probe.
Histology, Immunohistochemistry, and In Situ Hybridization
For histological study, samples of cardiac tissue were calibrated for the level of coronary blood flow as previously described14 19 20 and fixed in 2% paraformaldehyde. The samples were embedded in paraffin, sectioned, and stained with hematoxylin and eosin or hematoxylin and basic fuchsin. The latter stain was useful in identifying myocardial infarction after short periods of time.22 The monoclonal antibody to human MCP-1, LS27.10F7-2, was supplied by LeukoSite Corp and is the same antibody as used in blocking MCP-1 function in the accompanying article.13 A peroxidase-based detection system with diaminobenzidine as a substrate (Vector Laboratories) was used for antibody detection.
In Situ Hybridization in Tissue Samples
In situ hybridization was performed on 2% paraformaldehyde–embedded sections. After sectioning and deparaffinization, the tissue was incubated in 2×SSC buffer once for 5 minutes and again for 60 minutes, prehybridized with 100 μL prehybridization solution (50% formamide, 4×SSC, 1×Denhardt's reagent, 0.5 mg/mL salmon sperm DNA, 0.25 mg/mL yeast tRNA, and 10% dextran sulfate) for 1 hour at room temperature. The tissue was washed with 2×SSC buffer and then exposed to 30 μL hybridization buffer (prehybridization buffer with riboprobe [200 ng/mL]) overnight at 42°C. The slides were washed in 250 mL 2×SSC buffer while being shaken at room temperature with the buffer changed every 20 minutes. Washing continued after 1 hour with 250 mL 1×SSC buffer while the slides were shaken at room temperature with the buffer changed every 20 minutes. After 1 hour, washing continued with 0.5×SSC buffer (250 mL) at 37°C for 30 minutes, followed by 0.5×SSC buffer (250 mL) at room temperature while the slides were shaken for 30 minutes. A final wash with 0.1×SSC buffer was done for 30 minutes while the slides were shaken at room temperature. For immunological detection, an anti-digoxigenin antibody and nitro blue tetrazolium staining of an alkaline phosphatase reaction were used as previously described17 except that normal goat serum was substituted for sheep serum and Triton X-100 concentration was 0.2%. Stain incubations were adjusted by examination of the sections at frequent intervals until optimal staining was reached (2 hours).
In Situ Hybridization in Cells
The procedure for cells was identical to that for tissue, with the following modifications adapted from Panoskaltsis-Mortari and Bucy.23 After fixation in 3% paraformaldehyde, cells were placed on slides and baked. No prehybridization step was performed. After hybridization, cells were incubated with RNAse A (40 μg/mL) at 37°C for 30 minutes, followed by a wash in 2×SSC+50% formamide at 42°C to 50°C. All washes were performed for 5 to 10 minutes. Triton X-100 was not used for immunological detection.
Phages (≈5×105) were screened with 32P-labeled human MCP-1 cDNA probe with isolation of two unique clones, which were sequenced. Two clones were ligated to create a clone that contained the entire coding region. Fig 1⇓ shows the complete nucleotide sequence of canine MCP-1 cDNA. The cDNA for MCP-1 comprises 630 bp, with an open reading frame of 303 nucleotides that encodes for 101 amino acids. Fig 1⇓ also shows the deduced amino acid sequence of cMCP-1. There is 78% identity at the amino acid level compared with the human amino acid sequence.
Regulation of MCP-1 mRNA Expression in CJVECs by Postischemic Cardiac Lymph
Cardiac lymph was collected via cannulation of the cardiac lymphatic system. The ability of the lymph to stimulate MCP-1 mRNA was assessed. Lane 1 shows induction by TNF-α (30 μg/mL). There was no increase of message level with PBS control. Preischemic cardiac lymph induced a minimal signal. However, lymph collected 1 hour after reperfusion demonstrated striking induction of MCP-1 mRNA, which peaked in the first hour (Fig 2⇓).
Regulation of Canine MCP-1 in Ischemic and Reperfused Myocardium
The expression of MCP-1 mRNA in experimental coronary artery occlusion and reperfusion was assessed in ischemic and reperfused segments. Fig 3⇓ shows representative experiments of animals subjected to 1 hour of ischemia followed by 1 hour of reperfusion. The experiment on the right labeled “Infarct” clearly demonstrates a reduction in blood flow (lanes 4 through 6), as measured by labeled microspheres, and a significant induction of MCP-1 mRNA. In contrast, the animal labeled “No Infarct” (lanes 1 and 2), although instrumented and occluded, did not experience a significant reduction in blood flow, presumably because of collateral blood supply, and did not show any evidence of MCP-1 mRNA induction compared with a control segment from the same animal as well as the experiment in which occlusion led to significant ischemia.
To examine the induction of MCP-1 mRNA and its relationship to various intervals of reperfusion after 1 hour of coronary occlusion, a series of experiments were performed in segments from different dogs with comparable ischemia. A representative group of experiments is shown in Fig 4⇓. MCP-1 mRNA is present by 1 hour, achieves peak steady state by 3 hours, and persists at such levels for at least 48 hours.
To assess the role of reperfusion in the induction of MCP-1 mRNA, a series of experiments were performed comparing animals subjected to ischemia with and without reperfusion (Fig 5⇓). Ischemic segments that were not reperfused had minimal induction of MCP-1 mRNA compared with segments of similarly ischemic myocardium that were reperfused.
Immunohistochemistry: In Situ Hybridization
Immunohistochemical staining was done with the antibody LS27.10F7-2, which was used as a blocking antibody in the lymph chemotaxis studies described in the accompanying article.13 At 1 hour of reperfusion, no consistent staining was seen in either control (data not shown) or ischemic reperfused segments (Fig 6D⇓), even though there was significant monocyte accumulation within the first hour of reperfusion, as described in the accompanying article. At 3 hours, immunohistochemical staining of MCP-1 with the antibody was seen throughout the ischemic-reperfused myocardium (Fig 6B⇓) in the areas of intense leukocyte infiltration. MCP-1 staining was seen on the endothelium of the small veins (venules) and intrafascicular veins as well as on the infiltrating leukocytes (Fig 6B and 6C⇓⇓). Note that continuous endothelial staining is seen in a class of thin-walled veins (venules) of ≈20- to 70-μm diameter as they empty into a larger intrafascicular vein, whose endothelium is similarly stained (Fig 6B⇓). The endothelium of arterioles (Fig 6B⇓) was not stained in any section. No MCP-1 staining was present in nonischemic tissue (data not shown).
In contrast, in situ hybridization studies suggested that MCP-1 synthesis might be induced only in the endothelium of the 20- to 70-μm veins (venules). Fig 7A⇓, a low-power view, shows many images of these small veins, with the highest concentration seen in the subendocardial area. Higher-power views show these to be thin-walled veins that are uniformly stained in their endothelium (Fig 7C⇓). In contrast, the endothelium of larger veins and arterioles is not stained (Fig 7C⇓). Fig 7D⇓ shows that in situ hybridization for MCP-1 is prominent in venules in viable tissue bordering an area of intense contraction band necrosis. Fig 7E⇓ is a section taken serially from the same tissue block stained with hematoxylin and eosin, demonstrating the presence of extensive leukocyte infiltration. Thus, the failure to detect MCP-1 mRNA in leukocytes could not be explained by absence of leukocyte infiltration. To further investigate this, we examined leukocytes that were isolated from cardiac lymph and therefore have transmigrated the vascular endothelium and entered the lymph rather than remaining in tissue. Leukocytes in cardiac lymph collected between 60 and 120 minutes of reperfusion demonstrate marked induction of ICAM-1 mRNA but no induction of MCP-1 mRNA (Fig 8⇓). MCP-1 induction was not seen in any lymph-derived leukocytes between 0 and 300 minutes of reperfusion (data not shown).
The accelerated inflammation associated with reperfusion of the previously ischemic myocardium has been studied in great detail, and a potential role of inflammatory injury has been postulated as a cause of reperfusion injury.4 Paradoxically, substantial evidence suggests that reperfusion of the previously ischemic myocardium, even after myocardial salvage is no longer possible, also exerts a positive effect on ventricular healing in both animal models9 10 and clinical studies.2 The role of monocyte-derived phagocytic functions in ventricular healing has been postulated for many years9 10 ; the defective healing associated with methylprednisolone therapy5 6 may well be related to impairment of monocyte phagocytic cell function.
MCP-1 is a member of the C-C chemokine family that activates a specific subset of leukocytes. In addition to monocytes, MCP-1 activates T lymphocytes24 and is a potent activator of exocytosis in basophils and mast cells.25 26 MCP-1 production has been demonstrated in vascular endothelium,27 tissue monocytes,28 29 and macrophages25 ; however, many other cell types are potential sources for this cytokine.25 Evidence suggests that MCP-1 is not stored within granules and that its induction involves stimulation of de novo protein synthesis.25 In addition to stimulation by proinflammatory cytokines,25 MCP-1 induction has been associated with shear stress on human umbilical vein endothelium.27 An extensive discussion of cellular sources and inducers of chemokines has recently been published.26
The data in this report suggest rapid induction of MCP-1 mRNA in the ischemic and reperfused myocardium, appearing within the first hour and peaking by the third hour of reperfusion. MCP-1 mRNA levels remained high throughout the first 48 hours of reperfusion and are found exclusively in previously ischemic segments.
Several important features of MCP-1 induction emerge. The induction of MCP-1 mRNA appearing in early reperfusion seems to require reperfusion of the previously ischemic myocardium. Both human recombinant TNF-α and postischemic cardiac lymph induce formation of MCP-1 mRNA in cultured dog vascular endothelial cells. The MCP-1–inducing activity in cardiac lymph appears to peak in the first hour of reperfusion. Although immunocytochemistry shows MCP-1 staining of both endothelium and infiltrating cells, in situ hybridization studies suggest that the primary cell site of MCP-1 induction is the endothelium of the small veins (venules), which we have previously associated with extravascular and intravascular marginated neutrophils.17 19 30 Finally, immunohistochemical evidence suggesting that MCP-1 protein expression is not prominent until the start of the fourth hour is compatible with the functional data in the accompanying article.13 The immunohistochemical localization of MCP-1 on mononuclear cells at 3 hours is compatible with the observed role of MCP-1 in mononuclear cell chemotaxis mediated by the specific MCP-1 receptors that effect chemotaxis.25
The data suggest the elaboration of a soluble factor dependent on reperfusion that is capable of inducing MCP-1 gene transcription. Recent data from our laboratory31 demonstrate that tissue mast cells in the myocardium store preformed TNF-α and that mast cell degranulation appears to accompany reperfusion of the previously ischemic myocardium, with appearance of TNF-α activity in postischemic cardiac lymph, which might induce MCP-1 formation, as demonstrated in Fig 2⇑. Ongoing MCP-1 synthesis at the venular endothelial level would then play an important role in the transmigration of tissue monocytes into the infarcted myocardium. Of potential interest is the observation that MCP-1 might actually continue to stimulate its own synthesis by activating tissue mast cells.25
The unexpected finding in these studies was the observation that MCP-1 mRNA was found almost exclusively in the endothelium of small veins and that, even after 3 hours, when substantial monocyte transendothelial migration had occurred, there was very little evidence of MCP-1 mRNA in infiltrating leukocytes. This is in sharp contrast to what was expected from data derived in vitro suggesting that transendothelial migration alone induces MCP-1 in human monocytes28 and that adhesion of human monocytes to immobilized P-selectin facilitates monocyte MCP-1 synthesis.32 The reason for this discrepancy is not obvious from these data; in situ hybridization studies of these sections with riboprobes of similar size have demonstrated ICAM-1 mRNA in infiltrating mononuclear cells,17 suggesting that they are activated. In addition, leukocytes seen in cardiac lymph also demonstrate no induction of MCP-1 despite clear induction of ICAM-1 (Fig 8⇑). It is possible that the induction of cytokine synthesis in vivo is also under control of inhibitory cytokines that might suppress induction in specific cell populations; eg, in vitro experiments with IL-4 demonstrate its inhibition of IL-6 synthesis in activated human monocytes.33
This report and the accompanying article13 seek to examine the time course and factors that influence mononuclear cell influx and transendothelial migration on reperfusion of the previously ischemic myocardium. The accompanying article13 demonstrates that monocytes marginate within the first hour of reperfusion and are seen to migrate into the tissue extracellular space with a time course similar to that observed with neutrophils. Although early monocyte chemotactic activity in postischemic cardiac lymph could be ascribed to C5a and transforming growth factor-β1, by the end of the third hour, MCP-1 accounted for a substantial proportion of the monocyte chemotactic activity. This suggested that the ongoing monocyte influx into cardiac tissue involved a component that was at least in part dependent on MCP-1 and led to the studies described in this article.
The role of MCP-1 in pathological processes has not been studied extensively. Its role in phagocyte accumulation, vascular leakage, and hemorrhage associated with experimental IgA immune complex alveolitis34 and also a model of pulmonary granuloma formation in the rat have been suggested by the use of neutralizing monoclonal antibody studies.35 36 The studies reported here and the accompanying article represent the first demonstration that MCP-1 is an important factor in the course of the inflammatory reaction to reperfusion of the previously ischemic myocardium and may play a significant role in monocyte trafficking into the reperfused myocardium.
Selected Abbreviations and Acronyms
|CJVEC||=||canine jugular vein endothelial cell|
|ICAM-1||=||intercellular adhesion molecule-1|
|MCP-1||=||monocyte chemoattractant protein-1|
|TNF-α||=||recombinant human tumor necrosis factor-α|
This work was supported by NIH grant HL-42550, grants from the VA Merit Review System, and NIH grants NS-32583, AI-28071, and HL-41408 (Dr Rossen, Dr Birdsall), the American Heart Association (Dr Ballantyne), and The Methodist Hospital Foundation (Dr Kukielka). Dr Kumar was a research fellow of the American Thoracic Society. The authors wish to thank Concepcion Mata for her editorial assistance with the manuscript and Linda Schutze for her expert technical assistance.
Guest editor for this article was Judith L. Swain, MD, University of Pennsylvania, Philadelphia.
- Received April 14, 1996.
- Revision received September 19, 1996.
- Accepted September 30, 1996.
- Copyright © 1997 by American Heart Association
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