Signalling pathways of nitric oxide.
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Animals↗
Biomedical subjects
J Pfeilschifter
Publications and source records attributed to J Pfeilschifter.
Lipoteichoic acid induces delayed protection in the rat heart: A comparison with endotoxin.
Classic ischemic preconditioning transiently (30 to 120 minutes) protects the myocardium against subsequent lethal ischemia/reperfusion injury. After dissipation of this acute protection, a second window of protection (SWOP) appears 12 to 24 hours later; this SWOP lasts up to 3 days. Several triggers induce a SWOP, including brief repetitive cycles of coronary artery occlusion, rapid ventricular pacing, stimulation of adenosine A(1) receptors, and administration of wall fragments of Gram-negative bacteria, such as lipopolysaccharide (LPS). The aim of this study was to investigate whether lipoteichoic acid (LTA), a cell wall fragment of Gram-positive bacteria, can induce a SWOP in a rat model of left anterior descending coronary artery (LAD) occlusion (25 minutes) and reperfusion (2 hours). Thus, 166 male Wistar rats were pretreated (2 to 24 hours) with saline, LTA (1 mg/kg IP), or LPS (1 mg/kg IP) and subjected to LAD occlusion/reperfusion. Pretreatment with LTA or LPS for 16 hours led to a substantial, approximately 65%, reduction in infarct size and a reduction in the release of cardiac troponin T into the plasma. The dose of LTA used had no toxic effect (on any of the parameters studied), whereas the same dose of LPS caused a time-dependent activation of the coagulation system and liver injury. By use of RNase protection assays, it was determined that LPS caused a time-dependent induction of tumor necrosis factor-alpha, interleukin-1beta, and manganese superoxide dismutase mRNA content in the heart, whereas LTA failed to induce manganese superoxide dismutase. LPS also caused an upregulation of the expression of intercellular adhesion molecule-1 and P-selectin, whereas LTA downregulated these molecules and attenuated the accumulation of polymorphonuclear granulocytes caused by myocardial ischemia/reperfusion. This study demonstrates for the first time that pretreatment with LTA at 8 to 24 hours before myocardial ischemia significantly reduces (1) infarct size, (2) cardiac troponin T, and (3) the histological signs of tissue injury in rats subjected to LAD occlusion and reperfusion. The mechanism(s) underlying the observed cardioprotective effects of LTA warrants further investigation but is likely to be related to its ability to inhibit the interactions between the coronary vascular endothelium and polymorphonuclear granulocytes. Therefore, LTA represents a novel and promising agent capable of enhancing myocardial tolerance to ischemia/reperfusion injury.
Animals↗
Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair.
Wound-healing disorders are a therapeutic problem of extensive clinical importance. Leptin-deficient ob/ob mice are characterized by a severely delayed wound healing that has been explained by the mild diabetic phenotype of these animals. Here we demonstrate that systemically and topically supplemented leptin improved re-epithelialization of wounds in ob/ob mice. Leptin completely reversed the atrophied morphology of the migrating epithelial tongue observed at the wound margins of leptin-deficient animals into a well-organized hyperproliferative epithelium. Moreover, topically supplemented leptin accelerated normal wound-healing conditions in wild-type mice. As assessed by immunohistochemistry, proliferating keratinocytes located at the wound margins specifically expressed the leptin-receptor subtype ObRb during repair. Additionally, leptin mediated a mitogenic stimulus to the human keratinocyte cell line HaCaT and human primary keratinocytes in vitro. Therefore, leptin might represent an effective novel therapeutic factor to improve impaired wound-healing conditions.
Administration, Topical↗
Osteoporosis due to cancer treatment: pathogenesis and management.
Many therapeutic regimens in cancer treatment carry the risk of causing or favoring the development of osteoporosis. Therapies in which hypogonadism may occur are most relevant in this respect. Prompt hormone replacement therapy is indicated in these patients. In patients in whom this is undesirable because of a hormone-dependent tumor, the risk of osteoporosis should be assessed by means of osteodensitometry, and prophylactic or therapeutic measures should be instituted if necessary. Early intervention improves outcome because osteoporosis therapy is most effective in preventing deterioration of bone mass. There remains much uncertainty in assessing the risk of combination chemotherapy with regard to the development of osteoporosis. Negative effects on the skeleton have, however, been demonstrated for individual drugs, such as methotrexate and ifosfamide. Negative effects of the tumor itself on bone metabolism may aggravate the degree of osteoporosis. Detailed data and long-term experience to assess the risk are urgently needed in this area and constitute an important research topic for the coming years and decades. This review discusses the most prevalent mechanisms of osteoporosis caused by cancer treatment and outlines therapeutic strategies for the prevention and treatment of therapy-induced bone loss.
Antineoplastic Combined Chemotherapy Protocols↗
Regulation of interleukin-18 (IL-18) expression in keratinocytes (HaCaT): implications for early wound healing.
Keratinocytes display a high basal level expression of IL-18. Tumor necrosis factor-alpha (TNF-alpha) mediated a large decrease in IL-18 mRNA levels in the human keratinocyte cell line HaCaT, which was accompanied by a subsequent accumulation of IL-18 protein in the cell culture supernatants, which was shown to be biologically active. By contrast, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), respectively, strongly decreased IL-18 mRNA expression in HaCaT keratinocytes in the absence of IL-18 protein release from the cells. Notably, a pre-treatment of the cells with EGF, or TGF-alpha clearly attenuated TNF-alpha-induced IL-18 protein, release and bioactivity. For the in vivo situation of cutaneous wound repair, we observed an increase in IL-18 protein, 10 hours post-wounding, that closely correlated to infiltration of neutrophils which are known as producers of TNF-alpha. Our data suggest that bioactive IL-18 might be tightly counter-regulated by platelet- and neutrophil-derived factors at the onset of repair.
Blood Platelets↗
Lack of interferon-gamma production despite the presence of interleukin-18 during cutaneous wound healing.
BACKGROUND: Recently, we have reported a rapid and strong induction of interleukin-18 (IL-18) upon cutaneous injury in mice. In this paper, we investigated a possible role of IL-18 in triggering interferon-gamma (IFN-gamma) production at the wound site. MATERIALS AND METHODS: Expression of IFN-gamma during cutaneous wound healing was analyzed by RNase protection assay, Western blot, ELISA, and immunohistochemical techniques in a murine model of excisional skin repair. RESULTS: We could not detect any IFN-gamma mRNA and protein expression during normal skin repair. Additionally, impaired healing in the genetically diabetic db/db mouse, which was used as a model for a prolonged inflammatory phase of repair, was characterized by largely elevated levels of IL-18 during the late phase of repair and an absence of IFN-gamma. Western blot analysis for T-cell- and monocyte/macrophage-specific marker proteins (CD4, F4/80) clearly revealed the presence of these subsets of leukocytic cells at the wound site, that are known to produce IFN-gamma in response to IL-18. Furthermore, we provide evidence that the presence of transforming growth factor-beta1 (TGF-beta1) at the wound site might reflect a counterregulatory mechanism in IL-18-induced IFN-gamma production, as TGF-beta1 strongly suppressed IL-18/phytohaemagglutinin (PHA)-induced IFN-gamma production by peripheral blood mononuclear cells (PBMC) in vitro. CONCLUSIONS: Normal tissue regeneration processes after cutaneous injury were not dependent on the presence of IFN-gamma in vivo, and IL-18 must serve additional roles rather than inducing IFN-gamma during the healing process.
Animals↗
Modulation of cytokine-induced expression of secretory phospholipase A2-type IIA by protein kinase C in rat renal mesangial cells.
Renal mesangial cells express the 14 kDa secretory phospholipase A2-type IIA (sPLA2-IIA) in response to interleukin-1beta (IL-1beta). In order to understand the regulation of cytokine-induced sPLA2-IIA induction in more detail, we investigated whether phorbol ester-activated protein kinase C (PKC) has an influence on the IL-1beta-induced expression of sPLA2-IIA. We found that treatment of mesangial cells with the biologically active phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate inhibited IL-1beta induction of sPLA2-IIA mRNA, protein, and activity, whereas the inactive compound 4alpha-phorbol 12,13-didecanoate was without effect. An 8-hr pretreatment with PMA, which led to down-regulation of PKC-alpha and -delta isoenzymes, still inhibited sPLA2-IIA induction. Only after down-regulation of PKC-epsilon isoenzyme by 24-hr preincubation with PMA were we able to reconstitute the IL-1beta-induced sPLA2-IIA expression. Thrombin as a physiological activator of PKC in mesangial cells exerted similar effects as PMA and inhibited sPLA2-IIA expression. The selective PKC inhibitor calphostin C potentiated IL-1beta induction of sPLA2-IIA mRNA levels and partially reconstituted the thrombin-induced inhibition of sPLA2-IIA mRNA and activity. These data show that IL-1beta induction of sPLA2-IIA can be modulated by PKC and that the epsilon-isoenzyme of the PKC family is the most likely candidate mediating the suppression of cytokine-induced sPLA2-IIA expression in mesangial cells.
Animals↗
Age-associated changes in the stimulatory effect of transforming growth factor beta on human osteogenic colony formation.
Previous studies have indicated that the mitogenic responsiveness of human bone cells may change with age. In the present study, we examined whether aging affects the capacity of transforming growth factor beta (TGF-beta) to stimulate the colony formation of human osteoprogenitor cells. Outgrowths of bone cells from 98 iliac crest biopsies were plated at a density of 25 cells/cm2 and cultured for 3 weeks in the presence of 10% fetal calf serum. Approximately 5% of the plated cells gave rise to clonal colonies. TGF-beta (10(-11) M) significantly increased the estimated number of cells per colony. However, the stimulatory effect of TGF-beta significantly declined with donor age (r = -0.26, P = 0.01). Whereas TGF-beta raised the average number of cells per colony in cultures from donors below the age of 50 years by 136+/-50%, the average increase was only 43+/-16% in donors older than 60 years. These data raise the possibility that aging may be associated with a declining capacity of TGF-beta to enlarge the pool of bone cells that can be generated from a single human osteoblast progenitor cell.
Adult↗
Immunopharmacology: anti-inflammatory therapy targeting transcription factors.
Immunopharmacology is one of the most dynamic areas in pharmacology encompassing classical immunosuppressive drugs which reveal completely new clues concerning their mode of action as well as novel molecular biology approaches for treating inflammatory and autoimmune diseases, infections and cancer. This article focuses on transcription factors that regulate cell activities involved in immune and inflammatory cell responses and how traditional anti-inflammatory compounds such as glucocorticoids, cyclosporins, tacrolismus and salicylates interfere with the activation cascades triggering the transcription factors. Moreover, promising new initiatives for selective therapeutics including recombinant anti-inflammatory cytokines and proinflammatory cytokine antagonists, and gene therapy will be presented.
Anti-Inflammatory Agents↗
Nitric oxide stimulates chronic ceramide formation in glomerular endothelial cells.
Exposure of glomerular endothelial cells for 24 h to compounds releasing NO, including spermine-NO, MAHMA-NO, and S-nitroso-glutathione, results in a dose-dependent and delayed (after 24 h) increase in the lipid signaling molecule ceramide. This NO-induced stimulation occurs in a cGMP-independent fashion since the membrane-permeant cGMP analogue dibutyryl cGMP has no effect on chronic ceramide production. Short-term incubation of endothelial cells for 20 min reveals that NO and dibutyryl cGMP fail to stimulate an acute ceramide increase, whereas TNF-alpha, a well-known activator of sphingomyelinases, is able to acutely increase ceramide formation. Interestingly, N-oleoylethanolamine, an acidic ceramidase inhibitor, potentiates NO-induced chronic ceramide production, indicating that ceramide generation rather than ceramide metabolism is modulated by NO. Furthermore, NO-induced delayed ceramide formation is partially inhibited by the thiol-specific inhibitor iodoacetamide and the radical scavenger alpha-tocopherol, suggesting a regulatory role of thiol-containing enzymes and the involvement of a redox-sensitive mechanism. In addition, NO causes an increased DNA fragmentation in glomerular endothelial cells which is further enhanced by N-oleoylethanolamine and can be mimicked by exogenous ceramide. In summary, these results imply that ceramide represents an important mediator of NO-triggered chronic cell responses like apoptosis. Inhibition of ceramide synthesis may provide a new therapeutic approach to the treatment of pathological conditions involving increased NO formation.
Animals↗
Nitric oxide donors induce stress signaling via ceramide formation in rat renal mesangial cells.
Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1, 2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2-4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFalpha cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFalpha. Furthermore, in the presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNFalpha leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.
Animals↗
Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells.
Under conditions associated with local and systemic inflammation, mesangial cells and invading immune cells are likely to be responsible for the release of large amounts of nitric oxide (NO) in the glomerulus. To further define the mechanisms of NO action in the glomerulus, we attempted to identify genes which are regulated by NO in rat glomerular mesangial cells. We identified vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase (FLT-1) to be under the regulatory control of exogenously applied NO in these cells. Using S-nitroso-glutathione (GSNO) as an NO-donating agent, VEGF expression was strongly induced, whereas expression of its FLT-1 receptor simultaneously decreased. Expressional regulation of VEGF and FLT-1 mRNA was transient and occurred rapidly within 1-3 h after GSNO treatment. Expression of a second VEGF-specific receptor, fetal liver kinase-1 (FLK-1/KDR), could not be detected. The inflammatory cytokine interleukin-1beta mediated a moderate increase in VEGF expression after 24 h and had no influence on FLT-1 expression. In contrast, platelet-derived growth factor-BB and basic fibroblast growth factor had no effect on VEGF expression, but strongly induced FLT-1 mRNA levels. Obviously, there is a differential regulation of VEGF and its receptor FLT-1 by NO, cytokines and growth factors in rat mesangial cells.
Animals↗
Expression and release of chemokines associated with apoptotic cell death in human promonocytic U937 cells and peripheral blood mononuclear cells.
To characterize mechanisms which may determine the fate of apoptotic cells, we investigated chemokine expression in apoptotic promonocytic U937 cells or peripheral blood mononuclear cells (PBMC). Exposure of U937 cells to etoposide (VP-16) or the nitric oxide (NO) donor DETA-NO, both inducers of apoptosis in these cells, was associated with increased expression of the chemokines IL-8 and macrophage inflammatory protein-1 alpha. Up-regulation of IL-8 mRNA expression by VP-16 or DETA-NO was observed as early as 4 h or 6 h, respectively, after onset of treatment and was still detectable after 19 h of exposure. A serine protease inhibitor prevented both VP-16-induced apoptosis and release of IL-8, whereas inhibition of p38 MAP kinases reduced IL-8 secretion only. Moreover, we observed that incubation with 2-chlorodeoxyadenosine (CdA) up-regulated release of IL-8 from adherent PBMC in parallel to induction of apoptosis. In these cells a modest but significant induction of TNF-alpha release by CdA was also detected. In addition, CdA augmented release of IL-8 from whole blood cultures. By facilitating adequate recruitment of phagocytes to sites of cell death, stress-induced up-regulation of chemokines associated with apoptosis may contribute to mechanisms aiming at efficient removal of apoptotic cells.
Apoptosis↗
Changes in the concentration of insulin-like growth factor I and transforming growth factor beta1 in rat femoral bone during growth.
Our knowledge of the concentration of growth factors in growing bone is limited. In the present study, we examined the developmental changes in the concentrations of insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) in the rat femur between weanling and maturity. We show that during the rapid growth phase there is a continuous rise in bone matrix IGF-I and TGF-beta in all compartments of the femoral bone. The association between IGF-I and TGF-beta is not only temporal, but with few exceptions is also observed within the animals of each age class. These data support the hypothesis that IGF-I and TGF-beta play an important role in the growth-associated accumulation of bone mass.
Aging↗
Age-related changes in insulin-like growth factor I and II in human femoral cortical bone: lack of correlation with bone mass.
The concentration of insulin-like growth factor-I (IGF-I) in human cortical bone declines with age, but the relevance of this decline for cortical bone turnover and bone mass is unknown. In the present study, we simultaneously assessed the concentration of IGF-I and -II in cortical bone matrix and histomorphometric parameters of bone mass and bone turnover in 125 samples from the proximal human femur shaft. Bone width decreased by 27% and porosity increased by 100% in female cortical bone between the fourth and the ninth decade. Similar, but weaker, changes tended to occur in male cortical bone. The concentrations of both IGF species were correlated with the percentage of osteons undergoing bone remodeling. However, despite age-related decreases in both IGF species in men and in IGF-I in women, neither of the IGFs accounted for age-related or age-independent variability in cortical porosity or bone width. In conclusion, these data suggest that the local concentrations of IGF-I and -II are related to cortical bone turnover. In contrast, our study provides no evidence for a major role of bone matrix IGF-I and -II as determinants of cortical bone mass in elderly individuals. Whether other components of the IGF system may be stronger determinants of cortical bone loss remains to be determined.
Adult↗
Transforming growth factor beta (TGF-beta) levels in the conditioned media of human bone cells: relationship to donor age, bone volume, and concentration of TGF-beta in human bone matrix in vivo.
Transforming growth factor-beta (TGF-beta) is thought to play an important role in human bone remodeling. In the present study, we examined constitutive differences in TGF-beta levels in primary bone cell cultures from the iliac crest of 112 women, aged 28-79 years. TGF-beta1 was the major TGF-beta isoform in the conditioned media, as determined by neutralizing TGF-beta activity with specific antibodies against TGF-beta1-3 in the mink lung cell bioassay, and by enzyme-linked immunoassay (ELISA). TGF-beta1 levels in the conditioned media did not change with donor age. There was a lack of association between TGF-beta levels in vitro and the concentration of matrix-associated TGF-beta in vivo. TGF-beta1 levels failed to be associated with the local trabecular bone volume in the complete study population (r = +0.15, p = 0.16, n = 89). A significant association between TGF-beta1 levels and bone volume was present in premenopausal women (r = +0.39, p = 0.02, n = 33), but was largely accounted for by the two samples with the highest TGF-beta concentrations. In conclusion, our data suggest that TGF-beta1 is the major TGF-beta isoform produced by human bone cells in vitro, and that the constitutive secretion of TGF-beta by bone cells does not change with age. Whether constitutive differences in TGF-beta secretion may be a determinant of human bone mass remains to be clarified in further studies.
Adult↗
Cross-talk between group IIA-phospholipase A2 and inducible NO-synthase in rat renal mesangial cells.
Features of glomerulonephritis are expression of the inducible form of NO synthase (iNOS) as well as expression of the secretory group IIA-phospholipase A2 (sPLA2) in mesangial cells. Interleukin 1beta (IL-1beta) induces both enzymes with a similar time course resulting in an increase in nitrite production and sPLA2-IIA activity. In this study we investigated the relationship between the formation of NO and sPLA2-IIA induction in rat renal mesangial cells. Incubation of mesangial cells with the NO-donor, spermine-NONOate, for 24 h induced sPLA2-IIA mRNA expression and activity, whereas S-nitroso glutathione alone had only a small stimulatory effect. Stimulation of cells with IL-1beta caused a marked increase in sPLA2-IIA mRNA and activity that were potentiated 3 fold by both NO donors. Coincubation of cells with IL-1beta and the NOS inhibitor, L-N(G) monomethylarginine (L-NMMA), caused a dose-dependent inhibition of cytokine-induced sPLA2-IIA mRNA expression and activity. sPLA2-IIA activity was not stimulated by 8-bromo-cyclic GMP indicating that NO-induced sPLA2-IIA induction is independent of cyclic GMP-mediated signal transduction. These data show that NO contributes to the expression by cytokines of sPLA2-IIA and establishes a novel type of interaction between iNOS and sPLA2-IIA in mesangial cells. This cross-talk between inflammatory mediators may help to promote and sustain an inflammatory state in the kidney.
8-Bromo Cyclic Adenosine Monophosphate↗
Glucocorticoids potently block tumour necrosis factor-alpha- and lipopolysaccharide-induced apoptotic cell death in bovine glomerular endothelial cells upstream of caspase 3 activation.
1. Endothelial cell damage in glomeruli and kidney arterioles appears to play a pivotal role in glomerular inflammatory diseases. Glomerular endothelial cells, a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration, die by apoptosis in response to tumour necrosis factor-alpha (TNF-alpha), TNF-alpha/basic fibroblast growth factor (bFGF), TNF-alpha/cycloheximide, and bacterial lipopolysaccharide (LPS). Apoptotic cell death is characterized by extensive DNA cleavage, DNA ladder formation, and characteristic morphological alterations. 2. In search for apoptosis-preventing signals, we identified glucocorticoids as potent death preventing factors. Co-treatment of cells with 10 nM dexamethasone and TNF-alpha, TNF-alpha/bFGF, TNF-alpha/cycloheximide, or LPS blocked roughly 90% of apoptotic cell death in glomerular endothelial cells. 3. Similarly to dexamethasone (TNF-alpha- and LPS-induced apoptosis are prevented with IC50 values of 0.8 and 0.9 nM, respectively), other synthetic and natural forms of glucocorticoids, such as fluocinolone, prednisolone, hydrocortisone, and corticosterone potently inhibited cell death with IC50 values of 0.2, 6, 50 and 1000 nM, for TNF-alpha and 0.7, 8, 100 and 500 nM for LPS, respectively. 4. Apart from glucocorticoids, mineralocorticoids such as aldosterone also blocked TNF-alpha/LPS-induced apoptosis (IC50 approximately 500 nM for TNF-alpha and approximately 500 nM for LPS), whereas sex hormones, i. e. beta-estradiol and testosterone remained without effect. 5. The protective effect of glucocorticoids (and mineralocorticoids) required glucocorticoid receptor binding as it could be antagonized by the glucocorticoid receptor antagonist RU-486. Concerning TNF-alpha and LPS signal transduction, we found that dexamethasone efficiently prevented TNF-alpha- and LPS-induced activation of caspase-3-like proteases. Therefore, we postulate inhibitory mechanisms upstream of terminal death pathways.
Animals↗