Comparing different pneumoperitoneum (12 vs. 15 mmHg) pressures with cytokine analysis to evaluate clinical outcomes in patients undergoing robotic-assisted laparoscopic radical cystectomy and intracorporeal robotic urinary diversion.

Background: Robotic cystectomy is the mainstay surgical intervention for treatment-refractory nonmuscle-invasive and muscle-invasive bladder cancer. However, paralytic ileus may complicate the postoperative recovery and may be a consequence of an inflammatory response associated with transient gut ischaemia. We have therefore investigated clinical, operative and inflammatory biomarker associations between paralytic ileus in the context of robotic cystectomy and intracorporeal ileal conduit urinary diversion. Methods: Prospective consective patients referred for robotic cystectomy were consented and included in the study, while patients >75 years old and converted to open procedure were excluded. The pneumoperitoneum pressure (PP) for carbon dioxide insufflation required to perform the procedure efficiently and safely was recorded (12 or 15 mmHg). We also recorded the postoperative days patients passed flatus and stools, whether they developed ileus, as well as other standard clinical and demographic data. The expression of select proinflammatory and anti-inflammatory cytokines was determined by multiplex analysis using a cytometric bead array with changes in profiles correlated with the pressures applied and with the existence of an ileus. Results: Twenty-seven patients were recruited, but only 20 were used in the study with 10 patients in each PP group. Seven patients were excluded all of whom had an extracorporeal ileal conduit formation. There were differences in the 40-min shorter operative time and 1 day shorter length of stay, as well as passing flatus 1 day and stools 1.5 days earlier in the 12 mmHg compared with the 15 mmHg group. More patients had ileus in the 15 mmHg group vs 12 mmHg group (30% vs. 10.0%). These were not statistically significant. Similarly, there were no statistical differences in the expression of proinflammatory cytokines at the two different pressures or between patient groups, but there were outliers, with the median indicating nonsymmetrical distribution. By comparison, anti-inflammatory cytokines showed some significant differences between groups, with IL-6 and IL-10 showing elevated levels postsurgery. No statistical difference was observed between pressures or the existence of an ileus, but the maximum levels of IL-6 and IL-10 detected in some patients reflect a pressure difference. Conclusions: The initial findings of this novel scientific study indicated a higher risk of paralytic ileus postrobotic cystectomy and robotic intracorporeal urinary diversion when a higher pressure of 15 mmHg is used compared with 12 mmHg. Although further studies are required to establish the linkage between cytokine profile expression, pressure and ileus, our initial data reinforces the advantages of lower pressure robotic cystectomy and intracorporeal urinary diversion in patient outcomes.

The Impact of Semicarbazide Sensitive Amine Oxidase Activity on Rat Aortic Vascular Smooth Muscle Cells.

Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble- and membrane-bound transmembrane protein expressed in the vascular endothelial and in smooth muscle cells. In vascular endothelial cells, SSAO contributes to the development of atherosclerosis by mediating a leukocyte adhesion cascade; however, its contributory role in the development of atherosclerosis in VSMCs has not yet been fully explored. This study investigates SSAO enzymatic activity in VSMCs using methylamine and aminoacetone as model substrates. The study also addresses the mechanism by which SSAO catalytic activity causes vascular damage, and further evaluates the contribution of SSAO in oxidative stress formation in the vascular wall. SSAO demonstrated higher affinity for aminoacetone when compared to methylamine (Km = 12.08 µM vs. 65.35 µM). Aminoacetone- and methylamine-induced VSMCs death at concentrations of 50 & 1000 µM, and their cytotoxic effect, was reversed with 100 µM of the irreversible SSAO inhibitor MDL72527, which completely abolished cell death. Cytotoxic effects were also observed after 24 h of exposure to formaldehyde, methylglyoxal and H2O2. Enhanced cytotoxicity was detected after the simultaneous addition of formaldehyde and H2O2, as well as methylglyoxal and H2O2. The highest ROS production was observed in aminoacetone- and benzylamine-treated cells. MDL72527 abolished ROS in benzylamine-, methylamine- and aminoacetone-treated cells (**** p < 0.0001), while ßAPN demonstrated inhibitory potential only in benzylamine-treated cells (* p < 0.05). Treatment with benzylamine, methylamine and aminoacetone reduced the total GSH levels (**** p < 0.0001); the addition of MDL72527 and ßAPN failed to reverse this effect. Overall, a cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs where SSAO was identified as a key mediator in ROS formation. These findings could potentially associate SSAO activity with the early developing stages of atherosclerosis through oxidative stress formation and vascular damage.

Semicarbazide-Sensitive Amine Oxidase (SSAO) and Lysyl Oxidase (LOX) Association in Rat Aortic Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. ßAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). ßAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.

Serum cytokine levels as markers of paralytic ileus following robotic radical prostatectomy at different pneumoperitoneum pressures.

BACKGROUND: To evaluate intraoperative and postoperative cytokines in patients who underwent robotic prostatectomy (RP) at a pressure of 12 or 15 mm Hg, and the risk of postoperative ileus. MATERIALS AND METHODS: We presented the first series evaluating intraoperative and postoperative cytokines in patients undergoing RP at a pressure of 12 or 15 mm Hg by a single surgeon. Changes in cytokine concentrations were shown to correlate with surgical outcomes and pathological states. The study investigated the changes in cytokine concentrations (interferon-γ, tumor necrosis factor-α, interleukin-1ß [IL-1ß], IL-2, IL-4, IL-6, IL-12, and IL-17) at different pneumoperitoneum pressures and their potential role in the development of postoperative ileus. RESULTS: The data on 10 consecutive patients confirmed that a lower pneumoperitoneum pressure was associated with lower cytokine levels and a lower risk of ileus. There were increased levels of postoperative interferon-γ, tumor necrosis factor-α, IL-12p70, IL-1ß, IL-2, IL-4, and IL-17a at 15 mm Hg when compared to 12 mm Hg. CONCLUSIONS: The data indicated that lower pressure RP reduced intra-/postoperative cytokine levels confirming our hypothesis. Larger patient numbers are required to further validate this but the implications of this data will benefit not only urological patients but also other speciality patients undergoing minimally invasive surgery.

2-Oxothiazolidine-4-carboxylic acid inhibits vascular calcification via induction of glutathione synthesis.

Arterial medial calcification (AMC), the deposition of hydroxyapatite in the medial layer of the arteries, is a known risk factor for cardiovascular events. Oxidative stress is a known inducer of AMC and endogenous antioxidants, such as glutathione (GSH), may prevent calcification. GSH synthesis, however, can be limited by cysteine levels. Therefore, we assessed the effects of the cysteine prodrug 2-oxothiazolidine-4-carboxylic acid (OTC), on vascular smooth muscle cell (VSMC) calcification to ascertain its therapeutic potential. Human aortic VSMCs were cultured in basal or mineralising medium (1 mM calcium chloride/sodium phosphate) and treated with OTC (1-5 mM) for 7 days. Cell-based assays and western blot analysis were performed to assess cell differentiation and function. OTC inhibited calcification ≤90%, which was associated with increased ectonucleotide pyrophosphatase/phosphodiesterase activity, and reduced apoptosis. In calcifying cells, OTC downregulated protein expression of osteoblast markers (Runt-related transcription factor 2 and osteopontin), while maintaining expression of VSMC markers (smooth muscle protein 22α and α-smooth muscle actin). GSH levels were significantly reduced by 90% in VSMCs cultured in calcifying conditions, which was associated with declines in expression of gamma-glutamylcysteine synthetase and GSH synthetase. Treatment of calcifying cells with OTC blocked the reduction in expression of both enzymes and prevented the decline in GSH. This study shows OTC to be a potent and effective inhibitor of in vitro VSMC calcification. It appears to maintain GSH synthesis which may, in turn, prevent apoptosis and VSMCs gaining osteoblast-like characteristics. These findings may be of clinical relevance and raise the possibility that treatment with OTC could benefit patients susceptible to AMC.

Modulation of Macrophage Function by Lactobacillus-Conditioned Medium.

Probiotics are used as microbial food supplements for health and well-being. They are thought to have immunomodulatory effects although their exact physiological mechanism of action is not clear. This study investigated the influence of probiotic Lactobacillus rhamnosus GG conditioned media (LGG-CM) on macrophage phagocytosis of non-pathogenic Escherichia coli HfrC. The gentamicin protection assay was used to study the bacterial killing phases of phagocytosis. Macrophages co-incubated with E. coli for an hour allowed them to ingest bacteria and then the rate of E. coli killing was monitored for up to 300 min to determine the killing or digestion of the bacteria by recovering them from the macrophage lysate. We found that the LGG-CM significantly increased the bacterial killing by approximately 6-fold when compared with that of controls. By contrast, this killing process was found to be associated with enhanced free radical production via the activation of NADPH oxidase, stimulated by the LGG conditioned medium. We also found that the conditioned medium had small effect on nitric oxide (NO) generation, albeit to a lesser extent. This work suggests that LGG-CM may play an important role in suppressing the total microbial load within the macrophages and hence, the extent to which pro-inflammatory molecules such as free radicals and NO are generated. The modulation of inflammation-promoting signals by LGG-CM may be beneficial as it modulates bacterial killing, and thereby prevents any collateral damage to host.

Uremic serum-induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2.

Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22α, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P<0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P<0.001), serum phosphate (P=0.042), receptor activator of nuclear factor κappa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and ß-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P<0.01) in human SMCs and decreased SM22α expression (P<0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P<0.01). Both SM22α and Klotho expression decreased significantly (P<0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22α and Klotho.

Impaired endogenous fibrinolysis at high shear using a point-of-care test in STEMI is associated with alterations in clot architecture.

Impaired endogenous fibrinolysis is an adverse prognostic biomarker in acute coronary syndrome (ACS). Abnormally dense in vitro fibrin thrombi have been demonstrated in ACS patients and related to hypofibrinolysis using cumbersome, laboratory-based methods. We aimed to assess endogenous fibrinolysis using a point-of-care technique and relate this to clot architecture. From patients with ST-segment elevation myocardial infarction (STEMI), venous blood was drawn immediately on arrival to assess thrombotic status. Blood was assessed using the point-of-care Global Thrombosis Test which measures occlusive thrombus formation under high shear and subsequently endogenous fibrinolysis (lysis time, LT). Two samples per patient were run in parallel. In one channel, the measurement was allowed to proceed as normal. In the other, after occlusion, thrombus was extracted, washed, fixed in glutaraldehyde, dried, sputter-coated, and assessed using scanning electron microscope. Endogenous fibrinolysis was strongly associated fibrin fibre thickness (p = 0.0001). As LT increased (less efficient fibrinolysis), the fibrin network of the thrombus was significantly more compact and dense, with thinner fibrin fibres and smaller gaps. Fibrin fibre thickness correlated inversely with LT (r = - 0.89, p = 0.001). Adverse clot architecture in vitro is directly related to impaired endogenous fibrinolysis using a relatively new point-of-care technique in patients with STEMI. This may transform the relevance of fibrin clot architecture from an off-line laboratory association to being directly relevant to endogenous fibrinolysis at the patient bedside, which could be used as a near-patient test to guide prognosis and assess the effect of treatment.

Diabetes confers in vitro calcific potential on serum which associates with in vivo vascular calcification.

Although vascular calcification (VC) is prevalent in Type 2 diabetes mellitus (T2DM), underlying mechanisms remain unclear. Neither is it known whether T2DM confers calcific potential (CP) on serum, enabling it to induce VC outside the disease milieu. We, therefore, investigated the CP of serum from controls and subjects with T2DM with and without in vivo VC. Samples from 20 healthy controls and 44 age- and sex-matched patients with T2DM with modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) > 60 ml·min-1 were analysed for CP using rat aortic smooth muscle cells in vitro CT scans of femoral arteries identified individuals with in vivo calcification. Serum from subjects with T2DM revealed significantly greater CP than controls. This was further enhanced in the presence of in vivo VC. Addition of ß-glycerophosphate (ß-GP) plus CaCl2 increased the CP of T2DM serum but not of controls. Along with age, CP was an independent predictor of the presence of VC. In receiver operator curve (ROC) analysis, CP was a significant predictor of femoral arterial VC (C-statistic 0.70: P=0.009). The distribution of CP was bimodal around a cutoff of 100 nmoles of Ca2+ protein mg-1, with a higher proportion of Type 2 diabetes subjects with in vivo calcification (T2DM+) sera above the cutoff value. This group also showed elevated levels of osteoprotegerin (OPG) and matrix Gla protein (MGP). Diabetes confers CP on the serum which is enhanced by the presence of in vivo VC. The CP acquired may be dependent on levels of OPG and MGP. These findings may be clinically relevant for early identification of individuals at risk of VC and for informing therapeutic strategies.

Lactobacillus rhamnosus GG conditioned media modulates acute reactive oxygen species and nitric oxide in J774 murine macrophages.

Phagocytes such as macrophages are capable of detecting and killing pathogenic bacteria by producing reactive oxygen and nitrogen species. Formation of free radicals in macrophages may be regulated by probiotics or by factors released by probiotics but yet to be identified. Thus, studies were carried out to determine whether cell-free conditioned medium obtained from cultures of Lactobacillus rhamnosus GG (LGG-CM) regulate production of reactive oxygen species (ROS) and/or nitric oxide (NO) in macrophages. J774 macrophages in culture were loaded with either H2DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Free radical production was measured on a fluorescence microplate reader and changes were analysed by Cumulative sum (CuSum) calculations. Low concentration of LGG-CM (10% LGG-CM) or LPS did not cause any significant change in basal levels of ROS or NO production. In contrast, high concentration of LGG-CM (75% and 100%) significantly enhanced ROS generation but also significantly reduced NO level. These findings are novel and suggest for the first time that probiotics may release factors in culture which enhance ROS production and may additionally reduce deleterious effects associated with excessive nitrogen species by suppressing NO level. These events may account, in part, for the beneficial bactericidal and anti-inflammatory actions ascribed to probiotics and may be of clinical relevance.

G-quadruplex formation of FXYD1 pre-mRNA indicates the possibility of regulating expression of its protein product.

G-quadruplexes are higher-order nucleic acid structures formed of square-planar arrangements of four guanine bases held together by Hoogsteen-type hydrogen bonds. Stacks of guanine tetrads are stabilised by intercalating potassium ions. FXYD1 encodes for phospholemman, a regulatory subunit of the cardiac Na(+)/K(+)-ATPase. Computational sequence analysis of FXYD1 pre-mRNA predicted the formation of stable intramolecular G-quadruplexes in human and orthologue sequences. Multiple sequence alignment indicated that G-rich sequences are conserved in evolution suggesting a potential role of G-quadruplexes in FXYD1 gene expression. The existence of a non-functional alternative splicing product indicated that the G-quadruplex formation may control alternative splicing. Quadruplex formation of human and bovine oligonucleotides was confirmed in vitro by native polyacrylamide gel electrophoresis and intrinsic fluorescence emission spectroscopy. Taking together the evolutionary conservation of G-quadruplex forming sequences with the confirmation of G-quadruplex formation in vitro by two FXYD1 homologues the results point to a potential role of these structures in regulating the expression of FXYD1 and thus regulate indirectly the activity of the cardiac Na(+)/K(+)-ATPase.

Human uraemic serum displays calcific potential in vitro that increases with advancing chronic kidney disease.

Vascular calcification (VC) strongly correlates with declining renal function and contributes to the high morbidity and mortality of patients with CKD (chronic kidney disease). It is closely regulated by circulating factors but little is known about the capacity of serum from patients to induce calcification outside the disease setting, which we now define as the calcific potential of serum. We have therefore examined the ability of serum from age- and sex-matched subjects with and without advancing CKD to induce calcification of cultured SMCs (smooth muscle cells). Samples from patients with CKD induced significant calcification compared with controls. More importantly, samples from patients on haemodialysis induced significantly higher calcification than those with moderate or advanced CKD. The calcification induced by the latter two but not those on haemodialysis could be enhanced with calcium chloride and ß-GP (ß-glycerophosphate). A positive correlation was evident between measured serum creatinine, phosphate, PTH (parathyroid hormone), OPG (osteoprotegerin) and the degree of calcification in vitro. eGFR (estimated glomerular filtration rate), DBP (diastolic blood pressure), haemoglobin and serum albumin correlated negatively. Stepwise multivariate analysis of log-transformed calcific potential data highlighted serum creatinine, albumin and OPG as significant predictors, explaining approximately 50% of the variation. Thus, other regulators, either not investigated or as yet unidentified, may contribute to the calcification potential of serum in vitro. Furthermore, uraemic serum can induce graded calcification outside of the disease milieu that reflects the degree of kidney impairment in vivo. These findings could have important clinical relevance in terms of developing novel diagnostic and/or therapeutic strategies for subjects with CKD.

Post-transcriptional divergence in the regulation of CAT-2A, CAT-2B and iNOS expression by dexamethasone in vascular smooth muscle cells.

Upregulation of L-arginine transport by pro-inflammatory mediators is a widely reported phenomenon which accompanies the expression of the inducible nitric oxide synthase (iNOS) enzyme in various cells. Both processes require de novo protein synthesis which may be regulated differentially through diverging signalling pathways. This is particularly defined by observations that the glucocorticoid dexamethasone, acting potentially through NF-κB, selectively blocks the expression of iNOS whilst having little or no effect on transport; suggesting that this ubiquitous transcription factor may not be required for induced transporter activity. This notion is however controversial as is the suggestion that dexamethasone may regulate iNOS expression exclusively through NF-κB. Thus, to further understand the mechanisms that control these processes, we have examined the level at which dexamethasone acts, investigating whether this involves NF-κB and whether the latter selectively regulates iNOS induction. Our current data directly demonstrate that induced L: -arginine transport is critically dependent on the activation of NF-κB, and further confirmed its role in the induction of iNOS in rat aortic smooth muscle cells. More importantly, dexamethasone enhanced both iNOS and CAT gene expression but repressed iNOS protein with no noticeable effects on transporter function or indeed NF-κB activation. These novel and unexpected findings reflect the complex nature of the regulation of iNOS by glucocorticoids and prove, contrary to previous assumptions, that dexamethasone can regulate CAT gene expression despite failing to alter transporter function. Moreover, the effects of dexamethasone occur through a non-NF-κB-mediated action even though NF-κB is required for both processes.

Lysophosphatidic acid protects mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis.

Bone marrow-derived mesenchymal stem cells (MSCs) have shown great promise for cardiac repair. However, poor viability of transplanted MSCs within the ischemic heart has limited their therapeutic potential. Our previous studies have documented that hypoxia and serum deprivation (hypoxia/SD), induced MSCs apoptosis through the mitochondrial apoptotic pathway. Since serum lysophosphatidic acid (LPA) levels are known to be significantly elevated after acute myocardial infarction and that LPA enhanced survival of other cell systems, we embarked on determining whether LPA protects MSCs against hypoxia/SD-induced apoptosis. We have also investigated the potential mechanism(s) that may mediate such actions of LPA. All experiments were carried out on rat bone marrow MSCs. Apoptosis was induced by exposure of cells to hypoxia/SD in a sealed GENbox hypoxic chamber. Effects of LPA were investigated in the absence and presence of inhibitors that target either G(i)proteins, the mitogen activated protein kinases ERK1/2, or phosphoinositide 3-kinase (PI3K). The data obtained showed that hypoxia/SD-induced apoptosis was significantly attenuated by LPA through Gi-coupled LPA(1) receptors linked to the downstream ERK1/2 and PI3K/Akt signaling pathways that function in parallel. Additional studies have demonstrated that hypoxia/SD-induced activation of mitochondrial dysfunction was virtually abolished by LPA treatment and that inhibition of the LPA(1) receptor, Gi proteins, the PI3K/Akt pathway, or ERKs effectively reversed this protective action of LPA. Taken together, our findings indicate that LPA is a novel, potent survival factor for MSCs and this may prove to be of considerable therapeutic significance in terms of exploiting MSC-based therapy in the infracted myocardium.

Inhibition of angiogenic tubule formation and induction of apoptosis in human endothelial cells by the selective cyclooxygenase-2 inhibitor 5-bromo-2-(4-fluorophenyl)-3-(methylsulfonyl) thiophene (DuP-697).

There are indications that inhibitors of the cyclooxygenase-2 (COX-2) enzyme may cause inhibition of angiogenesis, proliferation of endothelial cells and induce apoptosis in cell systems. The concentrations of inhibitors required for such effects are however much higher than those needed to inhibit COX-2, suggesting that the latter may not be involved in these actions of the drugs. We have however generated data that strongly indicates a critical role for COX-2 suppression in the inhibition of angiogenesis and induction of apoptosis in human cultured umbilical vein endothelial cells (HUVECs) by the selective cyclooxygenase-2 (COX-2) inhibitor 5-bromo-2-(4-fluorophenyl)-3-(methylsulfonyl) thiophene (DuP-697). DuP-697 concentration-dependently inhibited prostaglandin E(2) (PGE(2)) production by HUVECs and at its known IC(50) for COX-2 inhibition of 10 nM inhibited basal and vascular endothelial cell growth factor (VEGF)-induced PGE(2) production by 80% and 85% respectively. DuP-697 also induced apoptosis as shown by FACs analysis, an increase in chromatin condensation and DNA laddering in HUVECS treated with the drug. Moreover, these effects were reversed by PGE(2) and by VEGF. In parallel studies, DuP-697 induced caspases 3, 8 and 9, with the caspase-3 specific inhibitor N-Acetyl-Asp-Glu-Val-Asp-al (DEVD-CHO) blocking the induction of apoptosis. Capillary-like tubule formation by HUVECs cultured on Matrigel was inhibited by DuP-697 and this inhibition was prevented by PGE(2) but not by DEVD-CHO. These results indicate that the induction of apoptosis and inhibition of tubule formation by DuP-697 involves the inhibition of COX-2 and that whereas the induction of apoptosis is caspase-dependent, the inhibition of tubule formation occurs through a caspase-independent mechanism.

Cannabinoid signalling in TNF-alpha induced IL-8 release.

The molecular events mediating the immunomodulatory properties of cannabinoids have remained largely unresolved. We have therefore investigated the molecular mechanism(s) through which R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl] pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl) methanone (WIN55212-2) modulate production of interleukin-8 (IL-8) in HT-29 cells. Release of IL-8 induced by tumor necrosis factor-alpha (TNF-alpha) was determined by enzyme-linked immunosorbent assay (ELISA). Changes in expression of inhibitory kappa B (IkappaB) were monitored by Western blotting and activation of nuclear factor-kappa B (NF-kappaB) was determined in electrophoretic mobility shift assay (EMSAs). TNF-alpha induced release of IL-8 was inhibited by WIN55212-2 which also blocked the degradation of IkappaB-alpha and activation of NF-kappaB induced by TNF-alpha. These data provide strong evidence that WIN55212-2 may modulate IL-8 release by negatively regulating the signaling cascade leading to the activation of NF-kappaB. These findings highlight a potential mechanism for the immunomodulatory properties of cannabinoids and contribute towards acquiring a clear understanding of the role of cannabinoids in inflammation.

Rate of transport of l-arginine is independent of the expression of inducible nitric oxide synthase in HEK 293 cells.

Expression of inducible nitric oxide synthase (iNOS) is generally accompanied by a parallel upregulation in l-arginine transport which is dependent, at least in part, on the synthesis of new carrier proteins. It is not clear however whether the induction of iNOS and its subsequent utilisation of l-arginine for NO synthesis contribute to the enhancement in l-arginine transport rates observed following induction of cells with pro-inflammatory mediators. To address this issue, we have transfected an iNOS construct in a pEGFP-N1 vector into HEK-293 cells and investigated the effects this has on l-arginine transport. The expression of iNOS through transfection resulted in the production of significant quantities of NO as detected by the standard Griess assay. Under these conditions, the transport of l-arginine was found to be unaltered, with rate of uptake being comparable in both transfected and non-transfected cells. Characterisation of the transporter(s) involved with uptake of l-arginine revealed features characteristic of the classical cationic amino acid transport system y(+). Further analysis of the expression profile of the cationic amino acid transporter (CAT) involved revealed the presence of transcripts for CAT-1 and CAT-2B. These data demonstrate that iNOS activity does not drive or enhance l-arginine transport despite the fact that HEK-293 cells transport l-arginine via the CATs, including CAT-2B which is thought to be critical for supply of substrate to iNOS.

Role of L-citrulline transport in nitric oxide synthesis in rat aortic smooth muscle cells activated with LPS and interferon-gamma.

(1) l-citrulline, a coproduct of nitric oxide synthase (NOS)-catalysed metabolism of l-arginine to nitric oxide (NO), is an important intermediate of the urea cycle and a precursor for l-arginine biosynthesis in vascular cells. (2) In the present study, we have examined the characteristics of l-citrulline transport, regulation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) and the ability of l-citrulline to sustain NO synthesis in rat cultured aortic smooth muscle cells. (3) l-citrulline transport was saturable with an apparent Km=1.6+/-0.2 mm and Vmax=5.9+/-0.6 pmol microg-1 protein min-1. Transport was pH-insensitive, partially Na+-dependent and markedly inhibited by substrates selective for amino-acid transport systems L and N but not by l-arginine or substrates for systems A, ASC, xc- or XAG. Moreover, transport was not altered in cells treated with LPS (100 microg ml-1) and IFN-gamma (50 U ml-1) for 0-24 h. (4) Unlike l-arginine, l-citrulline could not sustain maximal NO production in cells expressing iNOS. (5) Our findings provide the first evidence in vascular smooth muscle cells that l-citrulline transport is mediated via a low-affinity carrier with characteristics resembling systems L and N. Moreover, in l-arginine-deprived rat aortic smooth muscle cells, l-citrulline cannot sustain maximal NO release via iNOS.