cyclic-gmp and Burns

Topics: Animals; Blood Platelets; Blood Vessels; Burns; Chemical Phenomena; Chemistry; Chemotaxis; Collagen; Cyclic AMP; Cyclic GMP; Dermatitis; Dermatitis, Contact; Humans; In Vitro Techniques; Leukocytes; Prostaglandins; Rabbits; Radioimmunoassay; Rats; Skin

Staphylococcus aureus is a leading cause for morbidity and mortality associated with skin and burn wound infections. Therapeutic options for methicillin-resistant S. aureus (MRSA) have dwindled and therefore alternative treatments are urgently needed. In this study, the immuno-stimulating and anti-MRSA effects of cyclic di-guanosine monophosphate (c-di-GMP), a uniquely bacterial second messenger and immuno-modulator, were investigated in HaCaT human epidermal keratinocytes and a murine skin wound infection model.. Stimulation of HaCaT cells with 125 μM c-di-GMP for 12 h prior to MRSA challenge resulted in a 20-fold reduction in bacterial colonization compared with untreated control cells, which was not the result of a direct c-di-GMP toxic effect, since bacterial viability was not affected by this dose in the absence of HaCaT cells. C-di-GMP-stimulated or MRSA-challenged HaCaT cells displayed enhanced secretion of the antimicrobial peptides human β-defensin 1 (hBD-1), hBD-2, hBD-3 and LL-37, but for hBD1 and LL-37 the responses were additive in a c-di-GMP-dose-dependent manner. Secretion of the chemokines CXCL1 and CXCL8 was also elevated after stimulation of HaCaT cells with lower c-di-GMP doses and peaked at a dose of 5 μM. Finally, pre-treatment of mice with a 200 nmol dose of c-di-GMP 24 h before a challenge with MRSA in skin wound infection model resulted in a major reduction (up to 1,100-fold by day 2) in bacterial CFU counts recovered from challenged skin tissue sections compared PBS-treated control animals. Tissue sections displayed inflammatory cell infiltration and enhanced neutrophil influx in the c-di-GMP pre-treated animals, which might account for the reduced ability of MRSA to colonize c-di-GMP pre-treated mice.. These results demonstrate that c-di-GMP is a potent immuno-modulator that can stimulate anti-MRSA immune responses in vivo and might therefore be a suitable alternative prophylactic or therapeutic agent for MRSA skin or burn wound infections.

Topics: Adjuvants, Immunologic; Animals; Burns; Cyclic GMP; Disease Models, Animal; Humans; Immunity, Innate; Keratinocytes; Methicillin-Resistant Staphylococcus aureus; Mice; Staphylococcal Skin Infections

Burn-induced heart dysfunction is a key factor for patient mortality. However, the molecular mechanisms are not yet fully elucidated. This study sought to understand whether burn-induced heart dysfunction is associated with cardiac mitochondrial dysfunction and interruption of the PDE5A-cGMP-PKG pathway. Sixty percent total body surface area (TBSA) scald burned rats (±sildenafil) were used in this study. A transmission electron microscope (TEM), real-time qPCR, O2K-respirometer, and electron transport chain assays were used to characterized molecular function. Cardiac mitochondrial morphological shapes were disfigured with a decline in mitochondrial number, area, and size, resulting in deficiency of cardiac mitochondrial replication. Burn induced a decrease in all mitDNA encoded genes. State 3 oxygen consumption was significantly decreased. Mitochondrial complex I substrate-energized or complex II substrate-energized and both of respiratory control ratio (RCRs) were decreased after burn. All mitochondrial complex activity except complex II were decreased in the burn group, correlating with decreases in mitochondrial ATP and MnSOD activity. Sildenafil, a inhibitor of the PDE5A-cGMP-PKG pathway, preserved the mitochondrial structure, respiratory chain efficiency and energy status in cardiac tissue. Furthermore, sildenafil treatment significantly restored ADP-conjugated respiration in burned groups. In conclusion, cardiac mitochondrial damage contributes to burn-induced heart dysfunction via the PDE5A-cGMP-PKG pathway.

Topics: Animals; Burns; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 5; Male; Mitochondria, Heart; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Signal Transduction

Epidermal stem cells (ESCs) play a critical role in wound repair, but the mechanism underlying ESC proliferation is unclear. Here, we explored the effects of nitric oxide (NO) on ESC proliferation and the possible underlying mechanism.. The effect of NO (two NO donors, SNAP and spermine NONOate, were used) on cell proliferation was detected using cell proliferation and DNA synthesis assays. Thereafter, expression of FOXG1 and c-Myc induced by NO was determined by immunoblot analysis. pAdEasy-FOXG1 adenovirus and c-Myc siRNA plasmids were infected or transfected, respectively, into human ESCs to detect the effect of FOXG1 and c-Myc on NO-induced cell proliferation. Additionally, NO-induced ESC proliferation in vivo was detected by BrdU incorporation and a superficial second-degree mouse burn model. Moreover, the relationships among NO, FOXG1 and c-Myc were detected by western blotting, real-time PCR and dual luciferase assay.. NO exerted a biphasic effect on ESC proliferation, and 100 μM SNAP and 10 μM spermine NONOate were the optimal concentrations to promote cell proliferation. Additionally, NO-promoted human ESC proliferation was mediated by FOXG1 and c-Myc in vitro and vivo. Furthermore, NO regulated FOXG1 expression through cGMP signalling, and NO-induced transcription of c-Myc was regulated by FOXG1-mediated c-Myc promoter activity.. This study showed that the biphasic effect of NO on ESC proliferation as well as NO induced ESC proliferation were regulated by the cGMP/FOXG1/c-Myc signalling pathway, suggesting that NO may serve as a new disparate target for wound healing.

Topics: Adolescent; Animals; Burns; Cell Proliferation; Cells, Cultured; Child; Cyclic GMP; Epidermal Cells; Forkhead Transcription Factors; Humans; Male; Mice, Inbred C57BL; Nerve Tissue Proteins; Nitric Oxide; Proto-Oncogene Proteins c-myc; Signal Transduction; Stem Cells; Wound Healing; Young Adult

This study was conducted to demonstrate the burn-induced lung neutrophil deposition and damage in rats is affected by the nitric oxide (NO)-dependent downstream cGMP signaling. In experiment 1, 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ) was given (20 mg/kg i.p.) to specific pathogen-free Sprague-Dawley rats immediately postburn to suppress the guanylate cyclase (GC) activity. At 8 h after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine 123 (DHR 123) oxidation and lung tissues were harvested for myeloperoxidase (MPO) determination and histological studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. In experiment 2, Sodium nitroprusside (SNP) was given (2 mM, i.p.) to elevate cGMP levels and ODQ (20 mg/kg, i.p.) or methylene blue (100 microM, i.p.) or saline was given. The animals were sacrificed 4 h after injection and lung tissues were harvested for iNOS mRNA study. The MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased up to 2-fold, 4-fold, and 2.5-fold after burn. Inhibition of GC by ODQ administration significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 55%, 66%, and 53%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. The protective effects of ODQ were comparable to the use of selective iNOS inhibitor as demonstrated previously. Furthermore, ODQ decreased the burn or SNP-induced iNOS mRNA levels at 4 h after burn. These findings suggest that burn-induced lung dysfunction is mediated by the NO/cGMP system because it is abolished by application of either iNOS inhibitor or GC inhibitor. Also, the beneficial effect of ODQ is partly due to the attenuation of burn-induced iNOS expression by GC inhibition.

Topics: Animals; Base Sequence; Burns; Cyclic GMP; Enzyme Inhibitors; Female; Gene Expression; Guanylate Cyclase; Lung; Lung Injury; Male; Methylene Blue; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroprusside; Oxadiazoles; Peroxidase; Quinoxalines; Rats; Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction

Hypertrophic scarring remains the most disabling sequela for burn survivors. Little is known about its pathogenesis. Collagen accumulation, however, has been consistently observed in burn hypertrophic scars (HS).. We have studied collagen production in the dermal fibroblasts derived from HS, which has developed for 9 months to 2 years. Reconstructive surgery was performed to remove HS from which the fibroblasts were cultured. Similarly, the normal cells were grown from the patient's donor site (DS), which provided autografting to the HS site. Collagen production in HS and DS fibroblasts was compared and analyzed in minimal essential amino acid medium containing 5% fetal bovine serum with inclusion of L-ascorbic acid (100 microg/mL) and beta-aminopropoinitrile (100 microg/mL) by monitoring a 20-h [3H]proline incorporation into bacterial collagenase III-digestible protein in the conditioned media.. We failed to detect any significant difference in collagen production in vitro between HS and DS. Irrespective of the fibroblasts from HS or DS, collagen production was substantially stimulated by human recombinant transforming growth factor beta-1 (TGF-beta1) (20 ng/mL) by approximately 250% after a 3-day pretreatment. In contrast, sodium nitroprusside (SNP) at 100 microM exhibited significant suppression (68%), which was rescued by hemoglobin (10 microM). TGF-beta1 significantly decreased nitric oxide (NO) production by 55%. In contrast, NO level drastically increased by 350% following SNP treatment. Epidermal growth factor showed no effect on either collagen production or NO level. The linear regression analysis shows a significant inverse correlation (r = 0.72; p < 0.05) of NO level with collagen production, suggesting the involvement of NO signaling in the modulation of collagen production. Consistent with the notion, we further showed that N-nitro-L-arginine methyl ester (100 microM) caused a synergistic stimulation and an arrested inhibition of collagen production in the presence of TGFbeta-1 and SNP, respectively. 8-BrcGMP (300 microM) mimicked the NO inhibitory action, while methylene blue (50 microM) restored the collagen production which was inhibited by SNP. Moreover, 8-BrcGMP offset the stimulation of collagen production.. The dermal fibroblasts derived from HS were not different from normals with respect to collagen production and their responses to regulations. The inhibition of collagen production was achieved by a cGMP-dependent NO action. TGFbeta-1 inhibited NO/cGMP signaling to ensure its stimulatory effect on collagen production in the dermal fibroblasts.

Topics: Burns; Cells, Cultured; Cicatrix; Collagen; Cyclic GMP; Fibroblasts; Humans; Linear Models; Microscopy, Fluorescence; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroprusside; Proline; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

This study examined the effects of arginine supplement of fluid resuscitation from burn injury on cardiac contractile performance and bacterial translocation after a third-degree burn comprising 43% of the total body surface area in adult rats. Before burn injury, rats were instrumented to measure blood pressure; after burn (or sham injury), paired groups of sham-burned and burned rats were given vehicle (saline), L-arginine, D-arginine, or N-methyl-L-arginine (300 mg/kg in 0.3 ml of saline 30 min, 6 h, and 23 h postburn) plus fluid resuscitation; sham-burned rats received drug only. Twenty-four hours after burn trauma, hemodynamics were measured; the animals were then killed and randomly assigned to Langendorff heart studies or to studies examining translocation of gut bacteria. Burn rats treated with vehicle, D-arginine, or N-methyl-L-arginine had well-defined cardiocirculatory responses that included hypotension, tachycardia, respiratory compensation for metabolic acidosis, hypocalcemia, cardiac contractile depression, and significant bacterial translocation. Compared with values measured in vehicle-treated burn rats, L-arginine given after burn improved blood pressure, prevented tachycardia, reduced serum lactate levels, improved cardiac performance, and significantly reduced bacteria translocation, confirming that L-arginine administration after burn injury provided significant cardiac and gastrointestinal protection. Circulating neutrophil counts fell after burn trauma and serum glucagon levels rose, but these changes were not altered by pharmacological intervention. Our finding of significantly higher coronary perfusate guanosine 3',5'-cyclic monophosphate concentration in L-arginine-treated burn rats suggests that the beneficial effects of L-arginine were mediated by nitric oxide production.

Topics: Animals; Arginine; Bacterial Translocation; Burns; Coronary Circulation; Cyclic GMP; Fluid Therapy; Hemodynamics; Nitric Oxide; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley; Resuscitation; Stereoisomerism; Ventricular Function

We investigated nitric oxide and cyclic GMP production in myocardium early after burn injury in rats. Nitric oxide synthase activity was measured in cytosol from the left ventricular wall of burned rats. Cytosol from control group animals was shown to contain mainly Ca2+-dependent nitric oxide synthase (cNOS) with a small amount of Ca2+-independent nitric oxide synthase (iNOS). Following burn injury, there was a marked increase in iNOS activity with a peak at 8 h post-burn, however, myocardial cNOS activity was found to decline. Parallel to iNOS induction there was a significant increase in myocardial nitric oxide and cyclic GMP production. All these changes were alleviated by treatment of the rats with dexamethasone. Since increases in cyclic GMP levels in the heart were associated with reduced myocardial contractility, it is possible that enhanced production of nitric oxide by a Ca2+-independent NO synthase accounts, at least in part, for the depression of myocardial contractility seen in burn animals and patients.

Topics: Animals; Anti-Inflammatory Agents; Burns; Cyclic GMP; Dexamethasone; Disease Models, Animal; Male; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Organ Culture Techniques; Rats; Rats, Wistar; Reference Values; Spectrophotometry

This study examined the effects of burn injury on coronary endothelial function and coronary vascular reactivity. Adult rabbits were given a scald burn over 30% of the total body surface area (or sham burn for controls) and resuscitated with Ringer's lactate solution (4 ml/kg/% burn). Subgroups of burned (n = 6) and sham-burned (n = 6) animals were sacrificed at 2, 6, and 24 hr after injury; hearts were harvested and perfused. Changes in coronary perfusion pressure (CPP, mmHg) and coronary vascular resistance (CVR, mmHg/min) were determined at a constant preload and constant coronary flow rate. Changes in coronary endothelial function were determined by the ability of the endothelium to release cGMP as an indicator of nitric oxide production. Compared to values measured in sham burns, CPP and CVR progressively fell during the early postburn period but increased toward values measured in the sham burn group by 24 hr. Cyclic GMP, fmole/ml of coronary perfusate, was significantly lower in burned hearts (27 +/- 1) compared to values measured in effluents from sham burn hearts (310 +/- 40, P < 0.05). Alterations in coronary effluent cGMP levels after burn injury suggest that thermal injury disrupts coronary endothelial function, likely contributing to postburn changes in cardiac performance.

Topics: Acetylcholine; Animals; Burns; Coronary Vessels; Cyclic GMP; Endothelium, Vascular; In Vitro Techniques; Myocardial Contraction; Nitroprusside; Rabbits; Ventricular Dysfunction

Signal transduction systems (sts(s)) and acetylcholinesterase (AChE) levels in skeletal muscle distantly located from the site of large body surface area (BSA) burns are due to the systemic effects of burn trauma. Evaluating the guanylate and adenylate cyclase sts(s), by measuring adenosine 3':5'-phosphate (cyclic AMP) and guanosine 3':5'-phosphate (cyclic GMP) by radioimmunoassay with polymorphic forms of AChE demonstrated that the various systems (i.e., cyclic GMP and AChE) interact while under the duress of burn trauma. This trauma emanates from large skin burns (30-50% BSA) that have induced a chronic burn trauma response at postburn day 21. This study showed that a system, with a minimum of 2 components, regulated cyclic AMP levels. This paper provides insight into our current understanding of the effects of burn trauma on cellular signalling and discusses some of the potential implications of recent findings on long term rehabilitative care.

Topics: Acetylcholinesterase; Animals; Burns; Centrifugation, Density Gradient; Cyclic AMP; Cyclic GMP; Guanylate Cyclase; Male; Mice; Models, Biological; Multivariate Analysis; Muscle, Skeletal; Signal Transduction

To elucidate the relationship between thermal injury and production of nitric oxide (NO), we measured the levels of heart No2-/NO3- (the stable and products of NO) and GMP in rats with full thickness burn of 30% TBSA. A higher level of heart tissue NO2-/NO3- was observed from 3 to 24 hours after burn injury. Parallel to the changes of NO2-/NO3-, there were increases of tissue cGMP level and heart tissue water content in burned rats. L-NMMA, a specific inhibitor of NO synthetase (NOS), can block the rise of heart NO2-/NO3- and the enhancement of heart water content. The results indicate that the heart injury caused by burn injury may be associated with induction of NOS and formation of NO.

Topics: Animals; Burns; Cyclic GMP; Female; Male; Myocardium; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley

The lymphocyte response to Con A and IL-2 synthesis was clearly suppressed in rats after thermal injury. In burn rats, the cAMP content in lymphocytes was significantly increased, but cGMP level was not changed. The serum of burn rat could inhibit Con A-induced lymphocyte proliferation and IL-2 production. The burn rat serum could also increase intracellular cAMP concentration. Dynorphin A enhanced lymphocyte proliferation and IL-2 synthesis, increased cAMP level in lymphocytes. Naloxone could inhibit the action of dynorphin A. Moreover, ynorphin A could partly reversed suppression effect of burn serum on lymphocyte proliferation and suppressed the increase of intracellular cAMP content induced by burn and rat serum. Our results suggested that dynorphin A augmented immune function of burn rats, and the action of dynorphin A was mediated by cAMP pathway.

Topics: Adjuvants, Immunologic; Animals; Burns; Cyclic AMP; Cyclic GMP; Dynorphins; Interleukin-2; Lymphocyte Activation; Male; Peptide Fragments; Rats; Rats, Sprague-Dawley

1. This study tested the hypothesis that the systemic effects of burn include altered metabolic activity in the heart. Metabolic activity was studied by measuring alterations in cyclic nucleotide levels and protein concentrations in atrial and ventricular muscle in mice at 14 and 22 days after a 20% body surface area (BSA) burn. Thermal injury was produced on the dorsal surface of anesthetized male CD mice by immersion in water at 95 degrees C for 8 s. This resulted in a full-thickness, 3 degrees scald burn. In atrial and ventricular tissues, levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) were analyzed by 125I-radioimmunoassay. 2. The protein content (mg prot g-1 dry wt) increased in the atria. The cyclic AMP content (nmol g-1 dry wt) was significantly increased fourfold and ninefold at 14 and 22 days, respectively, in atria from burned animals compared to controls. The cyclic AMP/cyclic GMP ratios were similarly increased. 3. In the ventricle, the protein content and cyclic AMP levels were not altered, but the cyclic AMP/cyclic GMP ratios (nmol g-1 dry wt) were increased at both 14 and 22 days. These changes both in atria and ventricles were less prominent when cyclic nucleotide concentrations or ratios were expressed as pmol mg-1 protein. 4. The data confirm the hypothesis that a 20% BSA thermal injury evokes effects in sites remote from burn injury such as in the atria and ventricles. These effects include total body weight loss, elevated cyclic AMP, cyclic AMP/cyclic GMP ratios, and protein levels in the atria, and elevated cyclic AMP/cyclic GMP ratios in both atrial and ventricular tissues at 2 and 3 weeks after thermal injury. To prevent underestimation of cyclic nucleotide levels such changes should preferably be expressed on a prot g- dry weight basis.

Topics: Animals; Body Weight; Burns; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Heart Atria; Heart Ventricles; Male; Mice; Muscle Proteins; Myocardium

Topics: Animals; Burns; Cyclic AMP; Cyclic GMP; Female; Liver; Male; Myocardium; Rats; Rats, Inbred Strains

This study tested the hypothesis that a single hindlimb burn has both local and distant effects. Male CF1 anaesthetized mice were given a full thickness scald burn of 3 per cent total body surface area (BSA) by immersion of their left hindlimb in water at 95 degrees C for 5 s. Muscle tension was measured through twitch analysis. Levels of cyclic adenosine 3'-5' monophosphate (cAMP) and cyclic guanosine 3'-5' monophosphate (cGMP) were analysed by 125I-radioimmunoassay. Measurements were made in gastrocnemei of the ipsilateral burned and contralateral unburned limbs over a 28-day postburn period. Within 1 week the burned limb showed an increase in both tension and a 100-fold increase in levels of cAMP. However, by the end of the second week muscle tension in the burned limb dropped to one-seventh of control values despite persistence of high levels of cAMP. In contrast, the systemic effects were manifested in the unburned contralateral limb which showed tension to undergo a six-fold compensatory increase at the end of the second week with a 75-fold increase in cAMP. By the end of 4 weeks, tension levels of both burned and unburned limbs were attenuated to one-half control values indicating neuromuscular (NM) dysfunction. Nevertheless, cAMP levels remained elevated in both limbs. Levels of cGMP were reduced throughout the 4-week postburn period. Subsequent to the single hindlimb injury both ipsilateral and contralateral gastrocnemei muscles showed elevated levels of total protein content.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Burns; Cyclic AMP; Cyclic GMP; Disease Models, Animal; Hindlimb; Male; Mice; Mice, Inbred Strains; Muscle Proteins; Muscles; Neuromuscular Diseases; Nucleotides, Cyclic

Topics: Adolescent; Adult; Burns; Child; Child, Preschool; Cyclic AMP; Cyclic GMP; Female; Humans; Male; Middle Aged

Measurement of 24-h urinary cyclic AMP and cyclic GMP levels in 19 patients for up to nine days after thermal injury has revealed differences in cyclic nucleotide excretion patterns between "severe" and "mild to moderate" groups of burned patients, classified according to a predictive index of burn mortality. Cyclic AMP excretion fell significantly from a high initial level in the "severe" group, but showed no significant change in the "mild to moderate" group. Differences in cyclic AMP excretion between the two groups of patients were only significant on the second day following burn injury. There was no correlation between cyclic AMP output averaged for each patient over the first nine days and percentage body surface area of the burn or the predictive index. In contrast to the results for cyclic AMP excretion, cyclic GMP output rose significantly over the 9-day period in severely burned patients, while levels reached a plateau on days four to six for the moderate to mild group. When cyclic GMP excretion was averaged over the first nine days for each patient, a significant correlation with both percent body surface area of burn and the predictive index of burn mortality was found. The tissue source or sources which are responsible for increased cyclic GMP excretion are as yet unknown.

Topics: Adolescent; Adult; Aged; Burns; Child; Cyclic AMP; Cyclic GMP; Female; Humans; Male; Middle Aged; Time Factors