Endotoxin stimulated cytokine production in rat vascular smooth muscle cells.

Because inflammatory processes may promote the development of atherosclerosis, we examined the activation of cytokine genes in rat vascular smooth muscle cells in vitro after treatment with bacterial lipopolysaccharide (LPS). Interleukin-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha) mRNA increased in response to LPS. Activation of nuclear factor-kappaB (NF-kappaB) presumably results in NF-kappaB binding to regulatory regions of target genes and activating transcription. We therefore compared the kinetics of NF-kappaB activation, cytokine message production, and TNF-alpha secretion. Maximum active NF-kappaB was found at 30 min after the addition of LPS and decreased thereafter. Increased IL-6 mRNA was detected at 30 min, increased TNF-alpha mRNA at 60 min, and increased IL-1 mRNA at 120 min. Secretion of TNF-alpha was dependent on LPS concentration and was first detected 120 min after LPS addition. Aspirin, which has been shown to inhibit NF-kappaB activation and cytokine secretion in other cell types, did not inhibit NF-kappaB activation or TNF-alpha secretion. However, aspirin reduced the amount of both TNF-alpha and IL-6 mRNA present 30 min after LPS addition by half (P < 0.05).

Stimulation of beta-adrenergic receptors inhibits the release of tumor necrosis factor-alpha from the isolated rat heart.

OBJECTIVES: Beta-adrenergic receptor agonists such as isoproterenol inhibit production of tumor necrosis factor (TNF)-alpha in a number of cell types. Because the heart is a source of TNF-alpha, we hypothesized that isoproterenol would inhibit cardiac production of the cytokine. DESIGN: Analysis of cardiac release of TNF-alpha. SETTING: Medical research laboratory. SUBJECTS: Rats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: With the approval of the Institutional Animal Care and Use Committee, rats were anesthetized and hearts were removed and perfused. After 30 mins, bacterial lipopolysaccharide (LPS) with or without isoproterenol was infused for 60 mins. At 30, 60, 90, 120, and 150 mins, coronary flow was measured and coronary effluent was analyzed for TNF-alpha. Cardiac production of TNF-alpha was expressed as pg/min. Cyclic adenosine monophosphate (AMP) in the coronary effluent was measured. TNF-alpha messenger RNA was determined in ventricular tissue. After 30 mins, TNF-alpha was undetectable in the coronary effluent However, 60 mins after the initiation of LPS infusion, TNF-alpha release was 875+/-255 pg/min and increased to 2164+/-721 pg/min at 150 mins. Simultaneous infusion of isoproterenol with LPS stimulated cyclic AMP release and inhibited TNF-alpha production. For instance, at 60 and 150 mins, TNF-alpha release was 75+/-38 and 58+/-29 pg/min, respectively (p < .05 vs. LPS alone). Simultaneous infusion of isoproterenol with LPS blocked the induction of TNF-alpha messenger RNA by LPS. Isoproterenol, begun 30 mins after the initiation of LPS infusion, still suppressed LPS-stimulated TNF-alpha release by 95% at 150 mins. Similar results were obtained with norepinephrine. CONCLUSIONS: Activation of beta-adrenergic receptors inhibits cardiac TNF-alpha release. This implies that cytokine production by the heart is inhibited by the sympathetic nervous system. In heart failure, the cardiac response to the sympathetic nervous system is impaired. This impairment may play a role in the high plasma levels of TNF-alpha found in heart failure.

Isoproterenol inhibits bacterial lipopolysaccharide-stimulated release of tumor necrosis factor-alpha from human heart tissue.

Recent evidence suggests that inflammatory cytokines, particularly tumor necrosis factor alpha (TNF-alpha), may play a role in heart disease. Elevated plasma levels of the cytokine have been reported in congestive heart failure and severe angina and after myocardial infarction. The exact role of TNF-alpha in heart disease and how production is stimulated and regulated in the heart are current areas of investigation. Regarding regulation of production, isoproterenol elevates cyclic AMP and inhibits TNF-alpha release in macrophages. Therefore we hypothesized that stimulation of beta-adrenergic receptors of the sympathetic nervous system would inhibit release of the cytokine from heart tissue. With Institutional Review Board approval and patient consent atrial tissue was obtained during preparation for cardiac bypass. The tissue was divided into segments, placed in culture medium, and incubated for various times in the presence or absence of lipopolysaccharide (LPS) (20 microg/mL) and/or isoproterenol (1 microM). The medium was removed and analyzed for biologically active TNF-alpha by the L929 cell cytotoxicity assay. Tissue samples were weighed and TNF-alpha release was expressed as pg TNF-alpha/mg tissue. Initially, to determine the time course of release, measurements were made at 2, 5, 10, 15, 30, 60, 120, 180, and 360 minutes after the addition of LPS. Elevated TNF-alpha levels in the culture medium were reliably detected at 360 minutes after exposure to LPS. In atrial tissue obtained from seven patients TNF-alpha released into the culture medium at 360 minutes was 6 +/- 3 pg/mg tissue. In the presence of LPS, levels of the cytokine in the culture medium increased to 604 +/- 233 pg/mg tissue (P < 0.05 vs LPS alone). When isoproterenol and LPS were simultaneously added to the culture medium release of TNF-alpha was reduced by 87 per cent to 82 +/- 40 pg/mg tissue (P < 0.05 vs LPS alone). Our results show that activation of the beta-adrenergic receptor inhibits myocardial production of TNF-alpha. This finding suggests that the sympathetic nervous system inhibits production of the cytokine and that impaired sympathetic function in heart failure may play a role in the elevated levels of TNF-alpha.

A reactive oxygen-generating system activates nuclear factor-kappaB and releases tumor necrosis factor-alpha in coronary smooth muscle cells.

BACKGROUND: Recently we reported that bacterial lipopolysaccharide (LPS) stimulates release of tumor necrosis factor alpha (TNF-alpha) from porcine coronary arteries and smooth muscle cells cultured from those vessels. It has also been reported that plasma levels of TNF-alpha are elevated after myocardial infarction. Since it is known that the production of reactive oxygen intermediates (ROI) occurs during ischemia and ROI are suggested activators of the nuclear regulatory factor kappaB (NF-kappaB), we tested the hypothesis that release of TNF-alpha from smooth muscle cells could also be stimulated with a ROI-generating system. MATERIALS AND METHODS: Smooth muscle cells were isolated from porcine coronary arteries. Confluent cells in 48-well culture dishes were treated for 30 min with 0.003 units/ml xanthine oxidase (XO) and 2 mM hypoxanthine (HX) added to the culture medium. The medium was then removed and the cells were washed three times and fresh medium without HX-XO was added. Then, at 1, 3, and 6 h the medium was removed and analyzed for biologically active TNF-alpha. In other experiments, smooth muscle cells were treated with 20 micrograms/ml LPS for 6 h and aliquots of medium analyzed for TNF-alpha. Untreated cells served as controls. Data were analyzed by two-way ANOVA with repeated measures. Extracts of total cell protein were prepared and activation of NF-kappaB was determined by electrophoretic mobility shift assay. RESULTS: Treatment of cells with HX-XO stimulated release of TNF-alpha, which rose to a maximum of 17.5 +/- 1.7 units/mg cell protein at 6 h. This was significantly higher (P < 0. 05) than release stimulated by LPS (10.2 +/- 1.0 units/mg at 6 h) or TNF-alpha detected in the culture medium from untreated control cells (4.2 +/- 0.9 units/mg protein at 6 h). Both HX/XO and LPS activated NF-kappaB. CONCLUSIONS: These results support the conclusion that coronary smooth muscle cells are a potential source of TNF-alpha during events that are associated with formation of ROI such as myocardial ischemia.

Adhesion formation after intraperitoneal and extraperitoneal implantation of polypropylene mesh.

Polypropylene mesh is commonly used in open and laparoscopic hernia repairs. We tested the hypothesis that intra-abdominal adhesion formation secondary to polypropylene mesh is greater when mesh is placed in an intraperitoneal versus an extraperitoneal position. Fifty adult male rats underwent midline laparotomy with or without implantation of a nonabsorbable mesh. There were ten rats in each of the following five groups: EP-M, creation of an extraperitoneal pocket without mesh placement; EP+M, mesh placement in an extraperitoneal pocket; IP+M, intraperitoneal mesh; IT-M, creation of an abdominal wall ischemic defect without mesh placement; IT+M, ischemic defect plus mesh. Adhesion formation was graded on a scale of 0 to 5, 2 weeks after operation. All groups formed adhesions. Tissue injury or the placement of a mesh in an intraperitoneal position resulted in significantly more adhesions. An entirely extraperitoneal approach to mesh placement is needed to minimize adhesions after laparoscopic hernia repair.

Release of tumor necrosis factor-alpha from coronary smooth muscle: activation of NF-kappaB and inhibition by elevated cyclic AMP.

BACKGROUND: Evidence suggests that tumor necrosis factor-alpha (TNF-alpha) is involved in heart diseases such as atherosclerosis. We used porcine coronary arteries and smooth muscle cells cultured from these vessels to study the regulation of production of TNF-alpha. The aims were to determine if bacterial lipopolysaccharide (LPS) could stimulate production; if activation of the nuclear regulatory factor, NF-kappaB, was associated with production; and if intracellular cAMP regulates TNF-alpha in coronary vasculature through a mechanism involving NF-kappaB. MATERIAL AND METHODS: LPS was used to stimulate TNF-alpha production. Forskolin (FSK) and 8-Br-cAMP were added to tissue and cells in order to elevate intracellular cAMP. TNF-alpha release into the bathing medium was measured by the L929 cell cytotoxicity assay. Intracellular cAMP was determined by radioimmunoassay. NF-kappaB activation was determined in whole cell extracts by electrophoretic mobility shift assay. RESULTS: In segments of coronary arteries, LPS stimulated TNF-alpha release which increased with time to a maximum at 6 h (485 +/- 19 units/g tissue) and remained elevated at this level for 24 h. In contrast, the level of TNF-alpha measured at 24 h in medium from coronary tissue not exposed to LPS was 11.1 +/- 4.1 units/g tissue. In the presence of LPS, both FSK and 8-Br-cAMP significantly reduced TNF-alpha release. For instance at 6 h in the presence of LPS and FSK or 8-Br-cAMP, TNF-alpha was 126 +/- 24 and 71.6 +/- 22 units/g tissue, respectively (P < 0.05 vs LPS alone). Tissue levels of cAMP were significantly elevated in the presence of FSK. Similar results were obtained with smooth muscle cells cultured from the coronary arteries; i.e., LPS stimulated TNF-alpha release which was inhibited in a concentration-dependent manner by a rise in intracellular cAMP induced by FSK. In cultured cells release of TNF-alpha stimulated by LPS was associated with activation of NF-kappaB. Neither FSK nor 8-Br cAMP inhibited activation of NF-kappaB by LPS. CONCLUSIONS: Porcine coronary arteries produce TNF-alpha from a smooth muscle cell source. Production stimulated by LPS was inhibited by elevated intracellular cAMP and was associated with activation of NF-kappaB. However, activation of NF-kappaB was not inhibited by elevated cAMP, suggesting that the regulatory action of this cyclic nucleotide could lie downstream from activation of the TNF-alpha gene. These results support the view that coronary vessels can be a source of TNF-alpha possibly involved in heart disease.

Formation of abdominal adhesions is inhibited by antibodies to transforming growth factor-beta1.

Transforming growth factor-beta (TGF-beta) is an important factor in regulating the inflammatory response and the production of extracellular matrix by fibroblasts. These two processes are linked in the formation of fibrous adhesions after abdominal surgery. When the mesothelium is injured a fibrin strand is produced which is populated first by inflammatory cells then by fibroblasts which secrete extracellular matrix forming a permanent adhesion. TGF-beta promotes both chemotaxis of monocytes and the production of extracellular matrix by fibroblasts. We have used a model of abdominal adhesions in rats in which a circle of peritoneum is dissected and then sutured into place again. After 2 weeks the rats are euthanized and the adhesions are scored. Six groups of 10 rats each underwent this surgery. Group I served as the operative control. Group II was treated with saline which was injected immediately after surgery and on Days 1 and 2 after surgery (vehicle control). Using the same protocol with saline as vehicle, the other four groups of rats were treated with nonspecific IgG (150 microgram per day), anti-TGF-beta (panspecific, 167 microgram per day), anti-TGF-beta1 (67 microgram per day), or anti-TGF-beta2 (50 microgram per day). The rats injected with anti-TGF-beta1 had significantly lower adhesion scores (P < 0.05) than the controls. Rats injected with anti-TGF-beta2 or anti-TGF-beta (panspecific) did not differ significantly from the control saline-injected rats. The results indicate that specifically reducing levels of TGF-beta1 alone can be effective in preventing abdominal adhesions.

Effect of rat mesenchymal stem cells on development of abdominal adhesions after surgery.

One of the common and most serious side effects of abdominal surgery is the formation of adhesions within the peritoneal cavity during healing. Efforts to prevent adhesion formation have concentrated on inhibiting the inflammatory response, inhibiting the formation or encouraging the lysis of fibrin, and protection of the damaged serosal surface. We are interested in regenerating the serosal surface by providing a source of mesothelial progenitor cells. Rats were divided into groups of 10 each. Abdominal adhesions were created by removing a circle of peritoneum and suturing it back into place. Two weeks later the rats were euthanized and the adhesions scored on a scale of 0-5. A population of mesenchymal stem cells (MSCs) isolated from the skeletal muscle of neonatal rats was tested. The cells were grown in primary culture to expand the population and then trypsinized and frozen at -80 degrees C. They are then thawed and grown in secondary culture before use. The control group were injected with saline i.p. immediately after surgery. The experimental groups received (1) 1.4 X 10(6) MSCs, (2) 5 X 10(6) MSCs, (3) 7.5 X 10(6) dead MSCs, (4) 5 X 10(6) rat smooth muscle cells immediately post-op, and (5) 5 X 10(6) MSCs 4-6 hours after surgery. Only live MSCs given immediately after surgery by i.p. injection significantly decreased the adhesion scores of the rats (mean score of 3.5 vs 0.9). MSCs injected i.p. 4-6 hours after surgery actually increased the adhesion scores (3.5 vs 4.7), and rat smooth muscle cells injected i.p. immediately after surgery had no effect on adhesions. The exact mechanism of action of the MSCs is unknown at this time. However, we postulate that the MSCs have the capacity to differentiate into mesothelial cells capable of repopulating the injured mesothelium.

A population of cells isolated from rat heart capable of differentiating into several mesodermal phenotypes.

A population of stem cells has been isolated from embryonic avian and neonatal rat skeletal muscle. These cells differentiate into several mesodermal phenotypes in culture upon treatment with dexamethasone. This study reports the isolation of a similar population of stem cells from another mesodermal tissue, the heart. Hearts were excised from 3- to 5-day- old rats, minced, and treated with a collagenase-dispase solution. Single cells were collected by centrifugation, washed, and plated in dishes. The cells were grown to confluence, trypsinized, and frozen at -80 degrees C in 7.5% dimethylsulfoxide. After at least 24 hr, the cells were thawed and plated in 24-well plates and treated with media containing dexamethasone at concentrations of 10(-6)-10(-10) M for 4 weeks. Control cultures contained mononucleated cells with a stellate morphology. Treatment with dexamethasone resulted in the appearance of several mesodermal phenotypes. Bone and cartilage nodules were identified with von Kossa and Alcian blue staining respectively. Adipocytes were identified using Sudan black B stain. Smooth muscle cells were identified by an anti-smooth muscle alpha-actin antibody, and skeletal myotubes were stained with anti-myosin antibody. Large binuclear cells with obvious fibers were noted and stained with anti-desmin. These binuclear cells appeared in both the control and the dexamethasone-treated cultures and were tentatively identified as cardiomyocytes. These data strongly suggest the existence of a population of mesenchymal stem cells in neonatal rat heart.

A population of cells resident within embryonic and newborn rat skeletal muscle is capable of differentiating into multiple mesodermal phenotypes.

We have previously shown a population of putative mesenchymal stem cells in the connective tissue surrounding embryonic avian skeletal muscle. These cells differentiate into at least five recognizable phenotypes in culture: fibroblasts, chondrocytes, myotubes, osteoblasts, and adipocytes. We have now isolated a similar population of cells from fetal and newborn rat skeletal muscle. Cells from rat leg muscle were dissected, minced, and then enzymatically digested with a collagenase-dispase solution. The dissociated cells were plated and allowed to differentiate into two recognizable populations: myotubes and stellate mononucleated cells. The cells were then trypsinized, filtered through a 20 microm filter to remove the myotubes, frozen at -80 degrees C, then thawed and replated. In culture the cells maintained their stellate structure. However, under treatment with dexamethasone, a nonspecific differentiating agent, seven morphologic conditions emerged: cells with refractile vesicles that stained with Sudan black B (adipocytes), multinucleated cells that spontaneously contracted in culture and stained with an antibody to myosin (myotubes), round cells whose extracellular matrix stained with Alcian blue, pH 1.0 (chondrocytes), polygonal cells whose extracellular matrix stained with Von Kossa's stain (osteoblasts), cells with filaments that stained with an antibody to smooth muscle a-actin (smooth muscle cells), cells that incorporated acetylated low density lipoprotein (endothelial cells), and spindle-shaped cells that grew in a swirl pattern (fibroblasts). The initial population is tentatively classified as putative mesenchymal stem cells. The presence of these cells point to the existence of stem cells in the postembryonic mammal that could provide a basis for tissue regeneration as opposed to scar tissue formation during wound healing.

Inhibition of epidural scar formation after lumbar laminectomy in the rat.

STUDY DESIGN: An animal model of laminectomy in rats was used to study scar tissue formation around the spinal cord. Dexamethasone, in controlled-release form, was tested in this system for its ability to decrease fibrous tissue formation. OBJECTIVES: The results were evaluated to determine whether dexamethasone in a biodegradable controlled-release vehicle could be used to limit scar tissue formation around the spinal cord after laminectomy. SUMMARY OF BACKGROUND DATA: Steroids can delay the formation of scar tissue. Continued treatment with dexamethasone results in various unacceptable side effects. Use of biodegradable controlled-release vehicles to deliver drugs may allow for prolonged low-dose treatment, concentrated at the surgical site, thereby avoiding side effects. METHODS: Forty-four Sprague Dawley rats underwent laminectomies and were treated with dexamethasone in one of two controlled-release vehicles or with vehicle alone. After 4 weeks, the rats were killed and histologic sections prepared from the spines were examined and graded by a pathologist. In addition, the dexamethasone preparations were introduced into Hunt-Schilling wound chambers, which were implanted in rats. Four weeks after implantation, the wound chambers were removed and the tissue inside was assayed for DNA and protein content. RESULTS: Dexamethasone acetate (Decadron, MSD, West Point, PA) significantly reduced the density of the scar tissue undermining the laminas. Steroids embedded in polymer did not change the scar formation in the back, but did decrease protein and DNA values in wound chamber tissues. CONCLUSIONS: Long-term release of small amounts of steroid from the polymer poly-carboxy-phenoxypropane does not appear to reduce scar at laminectomy sites but does decrease the protein:DNA ratio in wound chambers. In contrast, Decadron does not significantly alter the biochemistry of wound chamber tissue but does reduce scar in the back.

Modulation of complement fixation and the phlogistic capacity of group A, B, and D streptococci by human lysozyme acting on their cell walls.

Streptococci and streptococcal cell wall fragments induce arthritis in rats, with the severity and duration depending on the capacity of the cells or cell fragments to resist degradation by tissue enzymes. Their phlogogenic effects are apparently related to their ability to activate the alternate complement pathway (ACP). The in vitro activation of the ACP by lysozyme-treated cells and cell walls of group A, B, and D streptococci suggests that both rat and human lysozyme can modulate this activity, i.e., increasing it, decreasing it, or doing both in that order. The effects of the lysozymes also correlated with the degree to which they can unmask the aminosugar-reducing groups detectable in a given amount of cell wall, which suggests that partial depolymerization of the cell wall is critical for ACP activation. The effects of mutanolysin and C phage lysin on ACP activation were found to be correlated with their action on streptococcal cell walls. Neuraminidase had relatively little effect on ACP activation by most streptococcal strains tested. We conclude that the participation of tissue enzymes, including but not necessarily limited to lysozyme, is an important determinant for the clinical arthritis induced by group A, B, or D streptococci. Experimental arthritis induced in rats with whole (or disrupted) streptococci may depend both on the capacities of the cell walls to activate the ACP and on the capacities of the host tissue enzymes to modulate this activation. Great severity and long durations of the disease were determined by the capacity of the enzymes to degrade cell wall antigens to a degree sufficient to ensure efficient activation of the ACP without completely degrading the material so that it no longer activates complement. In this model, the limited resistance of group B peptidoglycan to lysozyme was a critical pathogenic factor.

Toxicity of group B Streptococcus agalactiae in adult rats.

Several strains of group B Streptococcus agalactiae were found to be lethal for young adult rats. When bacteria were heat killed and then injected intraperitoneally into rats, rapid death (14 to 18 h) of the rats occurred, characterized by labored breathing, hemolyzed serum, hemoglobinuria, and subungual hemorrhages. Sections of tissues from these rats failed to reveal the cause of death. Rats injected with toxic or nontoxic strains of group B S. agalactiae had reduced numbers of circulating leukocytes and low serum C3 levels in comparison with those in control rats. The toxic strains of group B S. agalactiae induced dramatic decreases in platelet numbers, and in plasma fibrinogen levels as well, suggesting that the toxicity was due to disruption of the coagulation system. Rapid death in the absence of infection suggests that group B S. agalactiae may have a cell-associated toxin that induces these changes. Such a toxin may be a contributory factor in the high mortality rate associated with group B streptococcal infections of the human neonate.

Rat arthritis due to whole group B streptococci. Clinical and histopathologic features compared with groups A and D.

Heat-killed streptococci of Groups A, B, and D injected intraperitoneally into Sprague-Dawley rats induced arthritis. The histopathologic features of the arthritis were those of erosive synovitis. Early acute lesions were associated with deposits of streptococcal antigens. The serogroups and the physical state of the streptococci determined the incidence, the time of onset, the duration, and the severity of the disease, the severity being a blend of degree of inflammation, tendency to relapse, and occurrence of ankylosis. Whole Group A usually failed to induce arthritis. Group A disrupted with sonication regularly induced arthritis after a 24-hour latent period. The disease lasted over 60 days and caused ankylosis. Whole Group B regularly induced arthritis but only after a latent period of 6-8 days. The disease lasted over 40 days and caused ankylosed joints. With sonicated Group B a similar disease was induced, except that, as with sonicated Group A, the latent period was 24 hours. Whole Group D induced disease after a latent period of 48 hours. The arthritis lasted only 2 weeks and was transient. In contrast to its effects on Group A and B cocci, sonication of Group D abrogated its capacity to induce arthritis. It is postulated that for whole streptococci, in contrast to sonicated streptococci, arthritogenicity depends on the sensitivity of the cocci to initial processing in vivo. Processing may be partial digestion by enzymes of phagocytes. Cocci such as those of Group A that are insensitive to processing, injected whole, tend not to cause arthritis, but when they do cause disease, it is chronic. A coccus, such as one of Group D, that is very sensitive to processing produces a transient arthritis after a short latent period, while a coccus of intermediate sensitivity, such as one of Group B, induces disease only after a substantial latent period, and the disease is severe and chronic. The nature of processing remains to be determined.