The prostaglandin E2 receptor EP3 controls CC-chemokine ligand 2-mediated neuropathic pain induced by mechanical nerve damage.

Prostaglandin (PG) E2 is an important lipid mediator that is involved in several pathophysiological processes contributing to fever, inflammation, and pain. Previous studies have shown that early and continuous application of nonsteroidal anti-inflammatory drugs significantly reduces pain behavior in the spared nerve injury (SNI) model for trauma-induced neuropathic pain. However, the role of PGE2 and its receptors in the development and maintenance of neuropathic pain is incompletely understood but may help inform strategies for pain management. Here, we sought to define the nociceptive roles of the individual PGE2 receptors (EP1-4) in the SNI model using EP knockout mice. We found that PGE2 levels at the site of injury were increased and that the expression of the terminal synthase for PGE2, cytosolic PGE synthase was up-regulated in resident positive macrophages located within the damaged nerve. Only genetic deletion of the EP3 receptor affected nociceptive behavior and reduced the development of late-stage mechanical allodynia as well as recruitment of immune cells to the injured nerve. Importantly, EP3 activation induced the release of CC-chemokine ligand 2 (CCL2), and antagonists against the CCL2 receptor reduced mechanical allodynia in WT but not in EP3 knockout mice. We conclude that selective inhibition of EP3 might present a potential approach for reducing chronic neuropathic pain.

Characterization of the Micro-Environment of the Testis that Shapes the Phenotype and Function of Testicular Macrophages.

Macrophages are important in the activation of innate immune responses and in a tissue-specific manner in the maintenance of organ homeostasis. Testicular macrophages (TM), which reside in the testicular interstitial space, comprise the largest leukocyte population in the testes and are assumed to play a relevant function in maintaining testicular immune privilege. Numerous studies have indicated that the interstitial fluid (IF) surrounding the TM has immunosuppressive properties, which may influence the phenotype of TM. However, the identity of the immunosuppressive molecules present in the IF is poorly characterized. We show that the rat testicular IF shifted GM-CSF-induced M1 toward the M2 macrophage phenotype. IF-polarized M2 macrophages mimic the properties of TM, such as increased expression of CD163, high secretion of IL-10, and low secretion of TNF-α. In addition, IF-polarized macrophages display immunoregulatory functions by inducing expansion of immunosuppressive regulatory T cells. We further found that corticosterone was the principal immunosuppressive molecule present in the IF and that the glucocorticoid receptor is needed for induction of the testis-specific phenotype of TM. In addition, TM locally produce small amounts of corticosterone, which suppresses the basal expression of inflammatory genes as a means to render TM refractory to inflammatory stimuli. Taken together, these results suggest that the corticosterone present in the testicular environment shapes the immunosuppressive function and phenotype of TM and that this steroid may play an important role in the establishment and sustenance of the immune privilege of the testis.

LPA1 receptor-mediated thromboxane A2 release is responsible for lysophosphatidic acid-induced vascular smooth muscle contraction.

Lysophosphatidic acid (LPA) has been recognized recently as an endothelium-dependent vasodilator, but several lines of evidence indicate that it may also stimulate vascular smooth muscle cells (VSMCs), thereby contributing to vasoregulation and remodeling. In the present study, mRNA expression of all 6 LPA receptor genes was detected in murine aortic VSMCs, with the highest levels of LPA1, LPA2, LPA4, and LPA6 In endothelium-denuded thoracic aorta (TA) and abdominal aorta (AA) segments, 1-oleoyl-LPA and the LPA1-3 agonist VPC31143 induced dose-dependent vasoconstriction. VPC31143-induced AA contraction was sensitive to pertussis toxin (PTX), the LPA1&3 antagonist Ki16425, and genetic deletion of LPA1 but not that of LPA2 or inhibition of LPA3, by diacylglycerol pyrophosphate. Surprisingly, vasoconstriction was also diminished in vessels lacking cyclooxygenase-1 [COX1 knockout (KO)] or the thromboxane prostanoid (TP) receptor (TP KO). VPC31143 increased thromboxane A2 (TXA2) release from TA of wild-type, TP-KO, and LPA2-KO mice but not from LPA1-KO or COX1-KO mice, and PTX blocked this effect. Our findings indicate that LPA causes vasoconstriction in VSMCs, mediated by LPA1-, Gi-, and COX1-dependent autocrine/paracrine TXA2 release and consequent TP activation. We propose that this new-found interaction between the LPA/LPA1 and TXA2/TP pathways plays significant roles in vasoregulation, hemostasis, thrombosis, and vascular remodeling.-Dancs, P. T., Ruisanchez, E., Balogh, A., Panta, C. R., Miklós, Z., Nüsing, R. M., Aoki, J., Chun, J., Offermanns, S., Tigyi, G., Benyó, Z. LPA1 receptor-mediated thromboxane A2 release is responsible for lysophosphatidic acid-induced vascular smooth muscle contraction.

Salt supplementation ameliorates developmental kidney defects in COX-2-/- mice.

Deficiency of cyclooxygenase-2 (COX-2) activity in the early postnatal period causes impairment of kidney development leading to kidney insufficiency. We hypothesize that impaired NaCl reabsorption during the first days of life is a substantial cause for nephrogenic defects observed in COX-2-/- mice and that salt supplementation corrects these defects. Daily injections of NaCl (0.8 mg·g-1·day-1) for the first 10 days after birth ameliorated impaired kidney development in COX-2-/- pups resulting in an increase in glomerular size and fewer immature superficial glomeruli. However, impaired renal subcortical growth was not corrected. Increasing renal tubular flow by volume load or injections of KCl did not relieve the renal histomorphological damage. Administration of torsemide and spironolactone also affected nephrogenesis resulting in diminished glomeruli and cortical thinning. Treatment of COX-2-/- pups with NaCl/DOCA caused a stronger mitigation of glomerular size and induced a slight but significant growth of cortical tissue mass. After birth, renal mRNA expression of NHE3, NKCC2, ROMK, NCCT, ENaC, and Na+/K+-ATPase increased relative to postnatal day 2 in wild-type mice. However, in COX-2-/- mice, a significantly lower expression was observed for NCCT, whereas NaCl/DOCA treatment significantly increased NHE3 and ROMK expression. Long-term effects of postnatal NaCl/DOCA injections indicate improved kidney function with normalization of pathologically enhanced creatinine and urea plasma levels; also, albumin excretion was observed. In summary, we present evidence that salt supplementation during the COX-2-dependent time frame of nephrogenesis partly reverses renal morphological defects in COX-2-/- mice and improves kidney function.

Angiotensin II-AT1-receptor signaling is necessary for cyclooxygenase-2-dependent postnatal nephron generation.

Deletion of cyclooxygenase-2 (COX-2) causes impairment of postnatal kidney development. Here we tested whether the renin angiotensin system contributes to COX-2-dependent nephrogenesis in mice after birth and whether a rescue of impaired renal development and function in COX-2-/- mice was achievable. Plasma renin concentration in mouse pups showed a birth peak and a second peak around day P8 during the first 10 days post birth. Administration of the angiotensin II receptor AT1 antagonist telmisartan from day P1 to P3 did not result in cortical damage. However, telmisartan treatment from day P3 to P8, the critical time frame of renal COX-2 expression, led to hypoplastic glomeruli, a thinned subcapsular cortex and maturational arrest of superficial glomeruli quite similar to that observed in COX-2-/- mice. In contrast, AT2 receptor antagonist PD123319 was without any effect on renal development. Inhibition of the renin angiotensin system by aliskiren and enalapril caused similar glomerular defects as telmisartan. Administration of the AT1 receptor agonist L162313 to COX-2-/- pups improved kidney growth, ameliorated renal defects, but had no beneficial effect on reduced cortical mass. L162313 rescued impaired renal function by reducing serum urea and creatinine and mitigated pathologic albumin excretion. Moreover, glomerulosclerosis in the kidneys of COX-2-/- mice was reduced. Thus, angiotensin II-AT1-receptor signaling is necessary for COX-2-dependent normal postnatal nephrogenesis and maturation.

Activated Platelets Induce an Anti-Inflammatory Response of Monocytes/Macrophages through Cross-Regulation of PGE2 and Cytokines.

Platelets are well known for their role in hemostasis and are also increasingly recognized for their roles in the innate immune system during inflammation and their regulation of macrophage activation. Here, we aimed to study the influence of platelets on the production of inflammatory mediators by monocytes and macrophages. Analyzing cocultures of platelets and murine bone marrow-derived macrophages or human monocytes, we found that collagen-activated platelets release high amounts of prostaglandin E2 (PGE2) that leads to an increased interleukin- (IL-) 10 release and a decreased tumor necrosis factor (TNF) α secretion out of the monocytes or macrophages. Platelet PGE2 mediated the upregulation of IL-10 in both cell types via the PGE2 receptor EP2. Notably, PGE2-mediated IL-10 synthesis was also mediated by EP4 in murine macrophages. Inhibition of TNFα synthesis via EP2 and EP4, but not EP1, was mediated by IL-10, since blockade of the IL-10 receptor abolished the inhibitory effect of both receptors on TNFα release. This platelet-mediated cross-regulation between PGE2 and cytokines reveals one mechanism how monocytes and macrophages can attenuate excessive inflammatory responses induced by activated platelets in order to limit inflammatory processes.

Evidence that links loss of cyclooxygenase-2 with increased asymmetric dimethylarginine: novel explanation of cardiovascular side effects associated with anti-inflammatory drugs.

BACKGROUND: Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. METHODS AND RESULTS: Transcriptome analysis of wild-type and cyclooxygenase-2(-/-) mouse tissues revealed 1 gene altered in the heart and aorta, but >1000 genes altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-l-arginine. Cyclo-oxygenase-2(-/-) mice had increased plasma levels of ADMA and monomethyl-l-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide is cardio-protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage. CONCLUSIONS: We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction.

COX-2 gene dosage-dependent defects in kidney development.

Deletion of cyclooxygenase (COX)-2 causes impairment of kidney development, including hypothrophic glomeruli and cortical thinning. A critical role for COX-2 is seen 4-8 days postnatally. The present study was aimed at answering whether different COX-2 gene dosage and partial pharmacological COX-2 inhibition impairs kidney development. We studied kidney development in COX-2(+/+), COX-2(+/-), and COX-2(-/-) mice as well as in C57Bl6 mice treated postnatally with low (5 mg·kg(-1)·day(-1)) and high (10 mg·kg(-1)·day(-1)) doses of the selective COX-2 inhibitor SC-236. COX-2(+/-) mice exhibit impaired kidney development leading to reduced glomerular size but, in contrast to COX-2(-/-) mice, only marginal cortical thinning. Moreover, in COX-2(+/-) and COX-2(-/-) kidneys, juxtamedullary glomeruli, which develop in the very early stages of nephrogenesis, also showed a size reduction. In COX-2(+/-) kidneys at the age of 8 days, we observed significantly less expression of COX-2 mRNA and protein and less PGE2 and PGI2 synthetic activity compared with COX-2(+/+) kidneys. The renal defects in COX-2(-/-) and COX-2(+/-) kidneys could be mimicked by high and low doses of SC-236, respectively. In aged COX-2(+/-) kidneys, glomerulosclerosis was observed; however, in contrast to COX-2(-/-) kidneys, periglomerular fibrosis was absent. COX-2(+/-) mice showed signs of kidney insufficiency, demonstrated by enhanced serum creatinine levels, quite similar to COX-2(-/-) mice, but, in contrast, serum urea remained at the control level. In summary, function of both COX-2 gene alleles is absolutely necessary to ensure physiological development of the mouse kidney. Loss of one copy of the COX-2 gene or partial COX-2 inhibition is associated with distinct renal damage and reduced kidney function.

Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors.

Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP(3) prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP(3) receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP(3) receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP(3) receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP(3) prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP(3) receptor as a target to induce laxative effects.

Bioavailability and allergoprotective capacity of milk-associated conjugated linoleic acid in a murine model of allergic airway inflammation.

BACKGROUND: Cross-sectional epidemiological studies have demonstrated that farm milk from traditional farm settings possesses allergoprotective properties. Up to now, it has not been clarified which milk ingredient is responsible for protection against allergic diseases. As farm milk is rich in conjugated linoleic acids (CLA), it is hypothesized that this n-3 polyunsaturated fatty acid family contributes to the allergoprotective capacity of farm milk. We aim to prove this hypothesis in a murine model of allergic airway inflammation. METHODS: To prove the bioavailability and allergoprotective capacity of milk-associated CLA in a standardized protocol, milk batches that differed significantly in terms of their CLA content were spray dried and incorporated into a basic diet by substituting the regular sunflower fat fraction. Initially, the milk CLA uptake from the diet was monitored via measurement of the CLA content in plasma and erythrocyte membranes obtained from supplemented mice. To determine whether a milk CLA-enriched diet possesses allergoprotective properties, female Balb/c mice were fed the milk CLA-enriched diet ahead of sensitization and a challenge with ovalbumin (OVA) and the parameters of airway inflammation and eisosanoid pattern were measured. RESULTS: In animals, supplementation with a diet rich in milk CLA resulted in elevated CLA levels in plasma and erythrocyte membranes, indicating bioavailability of milk fatty acids. Though membrane-associated phospholipid patterns were affected by supplementation with milk CLA, this application neither reduced the hallmarks of allergic airway inflammation in sensitized and OVA-challenged mice nor modified the eiconsanoid pattern in the bronchoalveolar lavage fluid of these animals. CONCLUSION: Milk-associated CLA was not capable of preventing murine allergic airway inflammation in an animal model of OVA-induced allergic airway inflammation.

Deletion of cyclooxygenase-2 in the mouse increases arterial blood pressure with no impairment in renal NO production in response to chronic high salt intake.

Experiments were designed to test the hypothesis that cyclooxygenase-2 (COX-2) activity attenuates the blood pressure increase during high NaCl intake by stimulation of endothelial nitric oxide synthase (eNOS)-mediated NO synthesis in the kidney medulla. COX-2(-/-) (C57BL6) an COX-2(+/+) mice were fed a diet with 0.004% (low salt, LS) or 4% (high salt, HS) NaCl for 18 days. Arterial blood pressure was recorded continuously using indwelling catheters. Food and water intake and diuresis were measured in metabolic cages. Urine osmolality and excretion of electrolytes, cGMP, cAMP, and NOx were determined, as well as plasma NOx and cGMP. There was a significant dependence of blood pressure on salt intake and genotype: COX-2(-/-) exhibited higher blood pressure than COX-2(+/+) both on HS and LS intake. COX-2(+/+) littermates displayed an increase in blood pressure on HS versus LS (102.3 ± 1.1 mmHg vs. 91.9 ± 0.9 mmHg) day and night. The mice exhibited significant blood pressure increases during the awake phase (night) that were larger in COX-2(-/-) on HS diet compared with COX-2(+/+). Water intake, diuresis, Na(+), and osmolyte excretions and NOx and cGMP excretions were significantly and similarly elevated with HS in COX-2(-/-) and COX-2(+/+). In summary, C57BL6 mice exhibit a salt intake-dependent increase in arterial blood pressure with increased renal NO production. COX-2 activity has a general lowering effect on arterial blood pressure. COX-2 dampens NaCl-induced increases in arterial blood pressure in the awake phase. In conclusion, COX-2 activity attenuates the changes in nocturnal blood pressure during high salt intake, and COX-2 activity is not necessary for increased renal nitric oxide formation during elevated NaCl intake.

Temporal expression of the PGE2 synthetic system in the kidney is associated with the time frame of renal developmental vulnerability to cyclooxygenase-2 inhibition.

Pharmacological blockade of cyclooxygenase-2 (COX-2) causes impairment of kidney development. The present study was aimed at determining temporal expression pattern and activity of the PGE(2) synthetic pathway during postnatal nephrogenesis in mice and its association to the time window sensitive to COX-2 inhibition. During the first 10 days after birth, we observed transient induction of mRNA and protein for microsomal PGE synthase (mPGES)-1 between postnatal days 4 (P4) and P8, but not for mPGES-2 or cytosolic PGE synthase (cPGES). PGE(2) synthetic activity using arachidonic acid and PGH(2) as substrates and also urinary excretion of PGE(2) were enhanced during this time frame. In parallel to the PGE(2) system, COX-2 but not COX-1 expression was also transiently induced. Studying glomerulogenesis in EP receptor knockout mice revealed a reduction in glomerular size in EP1(-/-), EP2(-/-), and EP4(-/-) mice, supporting the developmental role of PGE(2). The most vulnerable time window to COX-2 inhibition by SC-236 was found closely related to the temporal expression of COX-2 and mPGES-1. The strongest effects of COX-2 inhibition were achieved following 8 days of drug administration. Similar developmental damage was caused by application of rofecoxib, but not by the COX-1-selective inhibitor SC-560. COX-2 inhibition starting after P10 has had no effect on the size of glomeruli or on the relative number of superficial glomeruli; however, growth of the renal cortex was significantly diminished, indicating the requirement of COX-2 activity after P10. Effects of COX-2 inhibition on renal cell differentiation and on renal fibrosis needed a prolonged time of exposition of at least 10 days. In conclusion, temporal expression of the PGE(2) synthetic system coincides with the most vulnerable age interval for the induction of irreversible renal abnormalities. We assume that mPGES-1 is coregulated with COX-2 for PGE(2) synthesis to orchestrate postnatal kidney development and growth.

The PGE(2)-EP4 receptor is necessary for stimulation of the renin-angiotensin-aldosterone system in response to low dietary salt intake in vivo.

Increased cyclooxygenase-2 (COX-2) expression and PGE(2) synthesis have been shown to be prerequisites for renal renin release after Na(+) deprivation. To answer the question of whether EP4 receptor type of PGE(2) mediates renin regulation under a low-salt diet, we examined renin regulation in EP4(+/+), EP4(-/-), and in wild-type mice treated with EP4 receptor antagonist. After 2 wk of a low-salt diet (0.02% wt/wt NaCl), EP4(+/+) mice showed diminished Na(+) excretion, unchanged K(+) excretion, and reduced Ca(2+) excretion. Diuresis and plasma electrolytes remained unchanged. EP4(-/-) exhibited a similar attenuation of Na(+) excretion; however, diuresis and K(+) excretion were enhanced, and plasma Na(+) concentration was higher, whereas plasma K(+) concentration was lower compared with control diet. There were no significant differences between EP4(+/+) and EP4(-/-) mice in blood pressure, creatinine clearance, and plasma antidiuretic hormone (ADH) concentration. Following salt restriction, plasma renin and aldosterone concentrations and kidney renin mRNA level rose significantly in EP4(+/+) but not in EP4(-/-) and in wild-type mice treated with EP4 antagonist ONO-AE3-208. In the latter two groups, the low-salt diet caused a significantly greater rise in PGE(2) excretion. Furthermore, mRNA expression for COX-2 and PGE(2) synthetic activity was significantly greater in EP4(-/-) than in EP4(+/+) mice. We conclude that low dietary salt intake induces expression of COX-2 followed by enhanced renal PGE(2) synthesis, which stimulates the renin-angiotensin-aldosterone system by activation of EP4 receptor. Most likely, defects at the step of EP4 receptor block negative feedback mechanisms on the renal COX system, leading to persistently high PGE(2) levels, diuresis, and K(+) loss.

Thromboxane-Induced α-CGRP Release from Peripheral Neurons Is an Essential Positive Feedback Loop in Capsaicin-Induced Neurogenic Inflammation.

α-CGRP is synthesized by sensory nerves in the dermis and its release can cause vasodilation and local inflammation. Its vasorelaxant effects are based on the direct activation of smooth muscle and endothelial cells, as well as the activation of mast cells causing the release of vasoactive and proinflammatory mediators. Here, we show that in the capsaicin model for neurogenic inflammation, capsaicin-induced edema formation is mediated by α-CGRP and mast cells, but is absent in thromboxane receptor-deficient mice. Capsaicin treatment of mice induced a thromboxane synthesis, which was mediated by α-CGRP and mast cells. Fittingly, α-CGRP induced thromboxane synthesis in mast cells and the thromboxane receptor agonist I-BOP caused edema formation independently of mast cells, suggesting that mast cells are the source of thromboxane. Most importantly, I-BOP-induced edema formation was mediated by α-CGRP and I-BOP was able to stimulate through calcineurin the α-CGRP release from peripheral neurons. Likewise, the signaling pathway, including α-CGRP, thromboxane receptor, and mast cells, also mediated capsaicin-induced mechanical hypersensitivity, a common symptom of capsaicin treatment. Taken together, the thromboxane-induced α-CGRP release from neurons forms a positive feedback loop causing prolonged α-CGRP release and edema formation during capsaicin-induced neurogenic inflammation.

GPR109A (PUMA-G/HM74A) mediates nicotinic acid-induced flushing.

Nicotinic acid (niacin) has long been used as an antidyslipidemic drug. Its special profile of actions, especially the rise in HDL-cholesterol levels induced by nicotinic acid, is unique among the currently available pharmacological tools to treat lipid disorders. Recently, a G-protein-coupled receptor, termed GPR109A (HM74A in humans, PUMA-G in mice), was described and shown to mediate the nicotinic acid-induced antilipolytic effects in adipocytes. One of the major problems of the pharmacotherapeutical use of nicotinic acid is a strong flushing response. This side effect, although harmless, strongly affects patient compliance. In the present study, we show that mice lacking PUMA-G did not show nicotinic acid-induced flushing. In addition, flushing in response to nicotinic acid was also abrogated in the absence of cyclooxygenase type 1, and mice lacking prostaglandin D(2) (PGD(2)) and prostaglandin E(2) (PGE(2)) receptors had reduced flushing responses. The mouse orthologue of GPR109A, PUMA-G, is highly expressed in macrophages and other immune cells, and transplantation of wild-type bone marrow into irradiated PUMA-G-deficient mice restored the nicotinic acid-induced flushing response. Our data clearly indicate that GPR109A mediates nicotinic acid-induced flushing and that this effect involves release of PGE(2) and PGD(2), most likely from immune cells of the skin.

Cyclooxygenases (COX-1 and COX-2) for tissue engineering of articular cartilage--from a developmental model to first results of tissue and scaffold expression.

Tissue engineering of articular cartilage remains an ongoing challenge. Since tissue regeneration recapitulates ontogenetic processes the growth plate can be regarded as an innovative model to target suitable signalling molecules and growth factors for the tissue engineering of cartilage. In the present study we analysed the expression of cyclooxygenases (COX) in a short-term chondrocyte culture in gelatin-based scaffolds and in articular cartilage of rats and compared it with that in the growth plate. Our results demonstrate the strong cellular expression of COX-1 but only a focal weak expression of COX-2 in the seeded scaffolds. Articular cartilage of rats expresses homogeneously COX-1 and COX-2 with the exception of the apical cell layer. Our findings indicate a functional role of COX in the metabolism of articular chondrocytes. The expression of COX in articular cartilage and in the seeded scaffolds opens interesting perspectives to improve the proliferation and differentiation of chondrocytes in scaffold materials by addition of specific receptor ligands of the COX system.

GPVI and Thromboxane Receptor on Platelets Promote Proinflammatory Macrophage Phenotypes during Cutaneous Inflammation.

Platelets are well known for their role in hemostasis but are also increasingly recognized for their supporting role in innate immune responses. Here, we studied the role of platelets in the development of peripheral inflammation and found that platelets colocalize with macrophages in the inflamed tissue outside of blood vessels in different animal models for cutaneous inflammation. Collagen-treatment of macrophages isolated from paws during zymosan-induced inflammation induced thromboxane synthesis through the platelet-expressed collagen receptor glycoprotein VI. Deletion of glycoprotein VI or its downstream effector thromboxane A2 receptor (TP) reduced zymosan-induced mechanical allodynia without altering macrophage recruitment or formation of macrophage/platelet complexes. Instead, macrophages in inflamed paws of glycoprotein VI- and TP-deficient mice exhibited an increased expression of anti-inflammatory markers and synthesized less proinflammatory mediators (prostaglandin E2 and IL6). TP expression on platelets was necessary to mediate increased prostaglandin E2 and IL6 synthesis, whereas TP expression on macrophages was sufficient to decrease the expression of the anti-inflammatory macrophage marker CD206, showing that TP activation on platelets and macrophages regulates different aspects of macrophage activation.

Efficiency of tazobactam/piperacillin in lethal peritonitis is enhanced after preconditioning of rats with O3/O2-pneumoperitoneum.

Insufflation of ozonized oxygen into the peritoneum (O3/O2-pneumoperitoneum [O3/O2-PP]) of rats reduced the lethality of peritonitis. We evaluated the prophylactic effect of O3/O2-PP combined with tazobactam/piperacillin (TZP) in polymicrobial lethal peritonitis. Wistar rats were conditioned by daily repeated insufflation of ozone for 5 days, and hematologic parameters were determined. Sepsis was induced by i.p. injection of cecal material derived from donor rats. Simultaneously, TZP was applied at a single dosage of 65 mg/kg or at two dosage schedules of 65 mg/kg each at an interval of 1 h. The conditioning effect of O3/O2-PP on the number of blood cells was measured before inoculation of bacteria. The mRNA levels of proinflammatory cytokine IL-lbeta and TNF-alpha were determined at 4 h post infection in spleen and liver by semiquantitative in situ hybridization analysis. Preconditioning of rats by O3/O2-PP enhanced the number of blood leukocytes and granulocytes and increased the survival rate of septic rats up to 33%. The combination of O3/O2-PP and TZP further enhanced the survival rate up to 93%. This effect was accompanied by a reduced amount of IL-1beta and TNF-alpha mRNA in spleen and liver. In contrast, in non-infected animals the combination of O3/O2-PP and TZP enhanced IL-1beta and TNF-alpha mRNA in the spleen and IL-1beta mRNA in liver when compared with TZP- and sham-treated controls. The preconditioning effect of O3/O2-PP seems to support the biological effectiveness of TZP by altering the immune status before and during the onset of sepsis. The combined therapy could be a simple, preoperative intervention for abdominal surgery to reduce postoperative morbidity and mortality.

Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor.

19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists.