Role Of iNOS Research Articles

A Role for iNOS in Erastin Mediated Reduction of P-Glycoprotein Transport Activity.

The blood-brain barrier is composed of both a physical barrier and an enzymatic barrier. Tight junction (TJ) proteins expressed between endothelial cells of brain capillaries provide the physical barrier to paracellular movement of ions and molecules to the brain, while luminal-facing efflux transporters enzymatically restrict the entry of blood-borne molecules from entering the brain. The expression and activity of ATP Binding Cassette transporters or "ABC" transporters in endothelial cells of the BBB and in human tumor cells are dynamically regulated by numerous signaling pathways. P-glycoprotein (P-gp), (ABCB1), is arguably the most studied transporter of the BBB, and in human cell lines. P-glycoprotein transport activity is rapidly inhibited by signaling pathways that call for the rapid production of nitric oxide (NO) from the inducible nitric oxide synthase enzyme, iNOS. This study investigated how nano-molar levels of the selective chemotherapeutic erastin affect the activity or expression of P-glycoprotein transporter in brain capillaries and in human tumor cell lines. We chose erastin because it signals to iNOS for NO production at low concentrations. Furthermore, erastin inhibits the cellular uptake of cystine through the XC- cystine/glutamate antiporter. Since previous reports indicate that NO production from iNOS can rapidly inhibit P-gp activity in tumor cells, we wondered if induction of iNOS by erastin could also rapidly reduce P-glycoprotein transport activity in brain endothelial cells and in human tumor cell lines. We show here that low concentrations of erastin (1 nM) can induce iNOS, inhibit the activity of P-glycoprotein, and reduce the intracellular uptake of cystine via the Xc- cystine/glutamate antiporter. Consistent with reduced P-glycoprotein activity in rat brain capillary endothelial cells, we show that human tumor cell lines exposed to erastin become more sensitive to cytotoxic substrates of P-glycoprotein.

Synergistic effect of arginine and Lactobacillus plantarum against potassium dichromate induced-acute liver and kidney injury in rats: Role of iNOS and TLR4/NF-κB signaling pathways.

Our study was conducted to evaluate the synergistic effect of arginine (ARG) and Lactobacillus plantarum against potassium dichromate (K2Cr2O7) induced-acute hepatic and kidney injury. Fifty male Wistar rats were divided into five groups. The control group received distilled water. The potassium dichromate group (PDC) received a single dose of PDC (20 mg/kg; SC). The arginine group (ARG) and Lactobacillus plantarum group received either daily doses of ARG (100 mg/kg, PO) or L. plantarum (109 CFU/ml, PO) for 14 days. The combination group (ARG+L. plantarum) received daily doses of ARG (100 mg/kg) with L. plantarum (109 CFU/ml), orally for 14 days, before induction of acute liver and kidney injury. Forty eight hours after the last dose of PDC, serum biochemical indices, oxidative stress biomarkers, pro-inflammatory cytokines, histopathological and immunohistochemical analysis were evaluated. Combining ARG with L. plantarum restored the levels of serum hepatic & kidney enzymes, hepatic & renal oxidative stress biomarkers, and TLR 4/ NF-κB signaling pathway. Furthermore, they succeeded in decreasing the expression of iNOS and ameliorate the hepatic and renal markers of apoptosis: Caspase-3, Bax, and Bcl2. This study depicts that combining ARG with L. plantarum exerted a new bacteriotherapy against hepatic and renal injury caused by PDC.

Neurobehavioral Toxicity Induced by Carbendazim in Rats and the Role of iNOS, Cox-2, and NF-κB Signalling Pathway.

Carbendazim (CBZ) is one of the most common fungicides used to fight plant fungal diseases, otherwise, it leaves residue on fruits, vegetables, and soil that contaminate the environment, water, animal, and human causing serious health problems. Several studies have reported the reproductive and endocrine pathological disorders induced by CBZ in several animal models, but little is known about its neurotoxicity. So that, the present study aimed to explain the possible mechanisms of CBZ induced neurotoxicity in rats. Sixty male Wistar rats were divided into 4 groups (n = 15). Group (1) received normal saline and was kept as the negative control group, whereas groups (2, 3, 4) received CBZ at 100, 300, 600mg/kg b.wt respectively. All rats received the aforementioned materials daily via oral gavage. Brain tissue samples were collected at 7, 14, 28days from the beginning of the experiment. CBZ induced oxidative stress damage manifested by increasing MDA levels and reducing the levels of TAC, GSH, CAT in some brain areas at 14 and 28days. There were extensive neuropathological alterations in the cerebrum, hippocampus, and cerebellum with strong caspase-3, iNOS, Cox-2 protein expressions mainly in rats receiving 600mg/kg CBZ at each time point. Moreover, upregulation of mRNA levels of NF-κB, TNF-α, IL-1B genes and downregulation of the transcript levels of both AchE and MAO genes were recorded in all CBZ receiving groups at 14 and 28days especially those receiving 600mg/kg CBZ. Our results concluded that CBZ induced dose- and time-dependent neurotoxicity via disturbance of oxidant/antioxidant balance and activation of NF-κB signaling pathway. We recommend reducing the uses of CBZ in agricultural and veterinary fields or finding other novel formulations to reduce its toxicity on non-target organisms and enhance its efficacy on the target organisms.

Effect of chronic intermittent hypoxia (CIH) on neuromuscular junctions and mitochondria in slow- and fast-twitch skeletal muscles of mice—the role of iNOS

BackgroundObstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles.MethodsMice were kept under 6 weeks of CIH (alternating 7% and 21% O2 fractions every 30 s, 8 h/day, 5 days/week) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy), and relevant transcripts (RT-qPCR). Besides wildtype (WT), we included inducible nitric oxide synthase knockout mice (iNOS−/−) to evaluate whether iNOS is protective or risk-mediating.ResultsIn WT soleus muscle, CIH vs. NOX reduced NMJ size (− 37.0%, p < 0.001) and length (− 25.0%, p < 0.05) together with fiber CSA of type IIa fibers (− 14%, p < 0.05) and increased centronucleated fiber fraction (p < 0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p < 0.001). Compared to WT, iNOS−/− similarly decreased NMJ area and length with NOX (− 55%, p < 0.001 and − 33%, p < 0.05, respectively) or with CIH (− 37%, p < 0.05 and − 29%, p < 0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p < 0.001) or swollen (> 6-fold, p < 0.001) mitochondria were observed with iNOS−/− vs. WT under NOX and similarly under CIH. Both, CIH- and iNOS−/− massively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) > 10-fold without changes in IL6 mRNA expression. Furthermore, inflammatory markers like CD68 (macrophages) and IL1β were significantly lower in CIH vs. NOX. None of these morphological alterations with CIH- or iNOS−/− were detected in the gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH.ConclusionCIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS−/− at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation without Il6 upregulation but accompanied by a decrease of macrophage density especially next to denervated endplates. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g., motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation.

The association between acute fatty liver disease and nitric oxide during malaria in pregnancy

BackgroundLiver disease is a common feature of malaria in pregnancy, but its pathogenesis remains unclear.MethodsTo understand the pathogenesis of liver disease during malaria in pregnancy, comparative proteomic analysis of the liver in a mouse model of malaria in pregnancy was performed.ResultsDecreased levels of mitochondrial and peroxisomal proteins were observed in the livers of pregnant mice infected with the lethal rodent malaria parasite Plasmodium berghei strain NK65. By contrast, increased levels of perilipin-2, amyloid A-1, and interferon (IFN)-γ signalling pathway-related proteins were observed in the livers of infected pregnant mice, suggesting that IFN-γ signalling may contribute to the development of liver disease during malaria in pregnancy. IFN-γ signalling is a potential trigger of inducible nitric oxide synthase (iNOS) expression. Liver disease associated with microvesicular fatty infiltration and elevated liver enzymes in pregnant wild-type mice infected with malaria parasites was improved by iNOS deficiency.ConclusionsIn this study, a causative role of iNOS in liver disease associated with microvesicular fatty infiltration during malaria in pregnancy was demonstrated. These findings provide important insight for understanding the role of iNOS-mediated metabolic responses and the pathogenesis of high-risk liver diseases in pregnancy, such as acute fatty liver.

Murine iNOS expression is essential for antifungal defenses in kidneys during disseminated cryptococcosis

Abstract C. neoformans, is one of the top 3 fungal pathogens, causing disseminated disease with high mortality in the absence of immunoprotecion. The protective Th1 immune response leads to M1 macrophage polarization that upregulates inducible nitric oxide synthase (iNOS) generating microbicidal nitric oxide. The role of iNOS has been studied in pulmonary defenses to C. neoformans, while its role in disseminated infection remains unknown. Here we study the effect of iNOS deletion in mouse model of disseminated cryptococcosis. The iNOS−/− and the WT, C57BL/6 mice were infected with 106 CFU of C. neoformans 52D via retro-orbital intravenous inoculation. Infected iNOS−/− mice showed significantly reduced survival times, however, they lacked significant increases in fungal burdens in the common infection sites (lung, spleen and CNS). Interestingly, iNOS deletion resulted in nearly 100-fold increase of the kidney fungal burden. Kidney histology revealed extensive lesions resulting in almost complete destruction of the kidney cortical area in iNOS−/− mice and a corresponding loss of kidney function was observed. The lack of fungal control was not due to a failure to recruit immune cells as iNOS−/− mice had increased kidney leukocyte recruitment as well as increases in monocytes and neutrophils. BMDMs from iNOS−/− mice were able to kill Cryptococcus despite a slight M2 bias seen in vivo. iNOS−/− mice had no defect in T-cell polarization, but a slight decrease in surface markers of activation. We conclude that iNOS expression is particularly important for local fungal control in kidneys, although its deletion does not induce a shift in a systemic immunophenotype in the iNOS−/− mice; rather it serves as an effector molecule in a local kidney environment.

Nicorandil mitigates folic acid-induced nephrotoxicity in mice: Role of iNOS and eNOS.

Folic acid (FA)-induced acute kidney injury (AKI) is a commonly used model in experimental animals for studying renal injury. This study aimed to investigate the probable protecting impact of nicorandil against FA-induced renal dysfunction. A mouse model was executed by a single injection of FA (250 mg/kg). Nicorandil was orally administrated in two doses (50 and 100 mg/kg) for 10 days. Nicorandil repressed the progression of FA-induced AKI as evidenced by the improvement of histopathological alterations and the substantial decrease of serum levels of creatinine, urea, blood urea nitrogen, malondialdehyde (MDA), and urinary protein levels. Moreover, nicorandil resulted in a profound reduction in oxidative stress as manifested by decreased MDA and increased reduced glutathioneand superoxide dismutasein renal tissue. Notably, nicorandil suppressed FA-induced inflammation; it reduced renal levels of nuclear factor-κB, tumor necrosis factor-α, and interleukin-6. Furthermore, nicorandil decreased renal levels of nitric oxide, inducible nitric oxide synthase, and increased endothelial nitric oxide synthase. Lastly, nicorandil efficiently decreased expression of the proapoptotic protein (Bax) and caspase 3. Nicorandil confers dose-dependent protection against FA-induced AKI by alleviating oxidative stress, inflammation besides modulating nitric oxide synthaseand reducing apoptosis.

Inducible nitric oxide synthase and systemic lupus erythematosus: a systematic review and meta-analysis

BackgroundThere is a growing body of evidences indicating iNOS has involved in the pathogenesis of SLE. However, the role of iNOS in SLE is inconsistency. This systematic review was designed to evaluate the association between iNOS and SLE.ResultsSix studies were included, reporting on a total of 277 patients with SLE. The meta-analysis showed that SLE patients had higher expression of iNOS at mRNA level than control subjects (SMD = 2.671, 95%CI = 0.446–4.897, z = 2.35, p = 0.019), and a similar trend was noted at the protein level (SMD = 3.602, 95%CI = 1.144–6.059, z = 2.87, p = 0.004) and positive rate of iNOS (OR = 9.515, 95%CI = 1.915–47.281, z = 2.76, p = 0.006) were significantly higher in SLE group compared with control group. No significant difference was observed on serum nitrite level between SLE patients and control subjects (SMD = 2.203, 95%CI = -0.386–4.793, z = 1.64, p = 0.095). The results did not modify from different sensitivity analysis, representing the robustness of this study. No significant publication bias was detected from Egger’s test.ConclusionsThere was a positive correlation between increasing iNOS and SLE. However, the source of iNOS is unknown. Besides NO pathway, other pathways also should be considered. More prospective random studies are needed in order to certify our results.

Increased inducible nitric oxide synthase (iNOS) expression in human myocardial infarction.

Increased inducible nitric oxide synthase (iNOS) expression has been reported in heart failure, cardiomyopathies, and arteriosclerosis. iNOS is expressed in the heart upon inflammatory stimuli and produces excessive amounts of nitric oxide (NO). The overproduction of NO is cytotoxic and involved in cardiovascular diseases. Furthermore, iNOS produces superoxide anion which proceeds with NO to the harmful oxidant peroxynitrite, causing oxidative stress in the heart. The aim of the study was to gain new insights into the role of iNOS in human myocardial infarction (MI) and its contribution to oxidative stress in the heart. Furthermore, we investigated the unaffected myocardium of the infarction hearts, to study if iNOS expression is increased, probably as an indicator for oxidative stress. Our results show a significant increase (p = 0.013) of the iNOS expression in the affected regions of MI hearts (n = 9) in comparison with healthy control hearts (n = 4). In the unaffected regions of MI hearts, an increase in the iNOS expression in some samples was found as well. Our study demonstrated the direct detection of iNOS mRNA in human myocardial tissue. The balance between beneficial and deleterious effects of iNOS may be particularly influenced by the presence or absence of concurrent oxidative stress.

Hepatocellular iNOS protects liver from ischemia/reperfusion injury through HSF1-dependent activation of HSP70

Although the role of inducible nitric oxide synthase (iNOS) in hepatic ischemia/reperfusion (I/R) injury remains controversial and confusing, with both harmful and beneficial effects in animal studies, the mechanism of these incongruous actions remains unclear. In the current study, we generated bone marrow chimeric mice with hepatocyte-restricted expression of iNOS. Chimeric mice and primary hepatocytes were subjected to I/R or anoxia/reoxygenation stimulation, respectively. The role of iNOS in liver I/R injury and the underlying molecular mechanisms were investigated. Hepatocyte-derived iNOS resulted in hepatoprotection from I/R injury, as well as in vitro experiments. Mechanistically, iNOS upregulates Heat shock protein (HSP) 70 by augmenting heat shock factor 1 (HSF1) binding to the HSP70 gene promoter. Importantly, inhibition of HSP70 partly reversed the iNOS overexpression-mediated hepatoprotection. The present findings demonstrate that hepatocellular iNOS protects from hepatic I/R injury through the HSF1-dependent activation of the HSP70. The upregulation of hepatocellular iNOS may offer a promising strategy for protecting against I/R injury.

Hepatic Lysosomal iNOS Activity Impairs Autophagy in Obesity.

Background & AimsThe lysosome is an acidic organelle that is important for maintaining cellular and metabolic homeostasis in hepatocytes. Lysosomal dysfunction and chronic inflammation coexist, and both contribute to obesity-associated hepatic insulin resistance. However, in the context of obesity, the interplay between inflammatory signals and hepatic lysosomal function remains largely unknown. Inducible nitric oxide synthase (iNOS) is a hallmark for inflammation, and is activated in obesity. The aim of this study is to understand the molecular link between iNOS-mediated lysosomal nitric oxide (NO) production, hepatic lysosomal function, and autophagy in the context of obesity-associated insulin resistance.MethodsThe role of iNOS in hepatic autophagy, as related to insulin and glucose homeostasis were studied in mice with diet-induced obesity (DIO). The effects and mechanisms of iNOS-mediated lysosomal NO production on lysosomal function and hepatic autophagy were studied in primary hepatocytes as well as in a mouse model of DIO.ResultsWe demonstrate that obesity promotes iNOS localization to the lysosome and decreases levels of lysosomal arginine, resulting in an accumulation of NO in hepatic lysosomes. This lysosomal NO production is attenuated by treatment with a NO scavenger, while co-overexpression of mTOR and a lysosomal arginine transporter (SLC38A9) enhances lysosomal NO production and suppresses autophagy. In addition, we show that deletion of iNOS ameliorates lysosomal nitrosative stress in the livers of DIO mice, promotes lysosomal biogenesis by activating transcription factor EB (TFEB), and enhances lysosomal function and autophagy. Lastly, deletion of iNOS in mice with DIO improves hepatic insulin sensitivity, which is diminished by suppression of TFEB or autophagy related 7 (Atg7).ConclusionsOur studies suggest that lysosomal iNOS-mediated NO signaling disrupts hepatic lysosomal function, contributing to obesity-associated defective hepatic autophagy and insulin resistance.

Investigation for effects of iNOS on biological function of chondrocytes in rats with post-traumatic osteoarthritis.

Inducible nitric oxide synthase (iNOS) is one rate-limiting enzyme in nitric oxide (NO) synthesis, and plays a role in mediating cell proliferation, apoptosis, and inflammation. Post-traumatic arthritis (PTA) is secondary after osteoarthritis (OA). This study thus established rat PTA model, on which the role of iNOS in PTA pathogenesis was investigated. Chondrocytes and synovial fluids were collected for comparing NO content and iNOS activity. In vitro cultured PTA rat chondrocytes were tested for expression of iNOS, matrix metallopeptidase 13 (MMP-13), and alpha-1 chain of type II collagen (COL2A1) expression with or without Interleukin 1β (IL-1β) treatment. NO content and iNOS activity in the supernatant were measured, followed by flow cytometry for Ki-67 expression and apoptosis. IL-1β treated cells were transfected with small interfere iNOS (si-iNOS), followed by expressional assay of iNOS, MMP-13 and Col2A1, activity assay of NO and iNOS, plus Ki-67 level and apoptosis. Compared to Sham group, PTA model rats had higher iNOS expression, NO content, and iNOS activity. IL-1β treatment remarkably elevated iNOS and MMP-13 expression in chondrocytes, it decreased COL2A1 expression, increased NO content and iNOS activity, whilst suppressed cell proliferation to facilitate apoptosis. Silencing of iNOS expression decreased iNOS or MMP-13 expression, increased COL2A1 expression, suppressed NO or iNOS content, potentiated proliferation and decreased apoptosis. iNOS expression and NO content were elevated in PTA rat chondrocyte. iNOS and NO production can facilitate chondrocyte apoptosis and matrix degradation, and suppress chondrocyte proliferation, thus playing a role in PTA pathogenesis.

INOS IMMUNOREACTIVITY IN THE OVARIES OF RATS FEEDING WITH MUSSEL (Mytilus galloprovincialis)

The ovary is an endocrine and exocrine gland that plays primary role in female development and reproductive activities. The seamen are collecting a lot of heavy metals by serving as filters in the seas. Since the last decades of the nineteenth century and until today, the study of the consequences of human exposure to heavy metals has risen as a central research area in the toxicological field. Among the group of metals with proven human toxicity aluminium (Al) and lead (Pb) are known to be highly neurotoxic. The Mediterranean mussel (Mytilus galloprovincialis) is a species of bivalve, a marine mollusc in the family Mytilidae. In our previous researches, we found Al, zinc (Zn) and iron (Fe) values were higher in mussels taken from Çamburnu region of the Dardanelles. In many tumors, inducible nitric oxide synthase (iNOS) expression is high, however, the role of iNOS during tumor development is very complex and quite perplexing, with both promoting and inhibiting actions having been described. The purpose of the study is to demonstrate the iNOS immunoreactivity in the ovarian tissues of rats which are fed with mussels that are collected from the Çamburnu region of the Dardanelles. The mussels given as food to the rats were removed from the Çamburnu region of the Dardanelles. Average 100±10 g weight were selected. After the beaks were overcooked, the meat broke off and the meat at 100 degrees was dried. Two groups of rats are included in the study, group 1 (n=6), control group fed with standard rat food, group 2 (n=6), 90% mussels and 10% standard rat food daily. To detect iNOS localization in the tissues, the LAB-SA Detection System was used. iNOS immunoreactivity was detected in the interstitial cell cytoplasm of the ovaries of rats fed with mussel. iNOS immunoreactivity was also observed in germ cell cytoplasm in some primordial follicles. There was statistically significant difference between the iNOS immunoreactivity of the interstitial cells in the ovarian parenchyma of the rats in the experimental and control groups (p&gt; 0.05).

INOS IMMUNOREACTIVITY IN THE OVARIES OF RATS FEEDING WITH MUSSEL (Mytilus galloprovincialis)

The ovary is an endocrine and exocrine gland that plays primary role in female development and reproductive activities. The seamen are collecting a lot of heavy metals by serving as filters in the seas. Since the last decades of the nineteenth century and until today, the study of the consequences of human exposure to heavy metals has risen as a central research area in the toxicological field. Among the group of metals with proven human toxicity aluminium (Al) and lead (Pb) are known to be highly neurotoxic. The Mediterranean mussel (Mytilus galloprovincialis) is a species of bivalve, a marine mollusc in the family Mytilidae. In our previous researches, we found Al, zinc (Zn) and iron (Fe) values were higher in mussels taken from Camburnu region of the Dardanelles. In many tumors, inducible nitric oxide synthase (iNOS) expression is high, however, the role of iNOS during tumor development is very complex and quite perplexing, with both promoting and inhibiting actions having been described. The purpose of the study is to demonstrate the iNOS immunoreactivity in the ovarian tissues of rats which are fed with mussels that are collected from the Camburnu region of the Dardanelles. The mussels given as food to the rats were removed from the Camburnu region of the Dardanelles. Average 100±10 g weight were selected. After the beaks were overcooked, the meat broke off and the meat at 100 degrees was dried. Two groups of rats are included in the study, group 1 (n=6), control group fed with standard rat food, group 2 (n=6), 90% mussels and 10% standard rat food daily. To detect iNOS localization in the tissues, the LAB-SA Detection System was used. iNOS immunoreactivity was detected in the interstitial cell cytoplasm of the ovaries of rats fed with mussel. iNOS immunoreactivity was also observed in germ cell cytoplasm in some primordial follicles. There was statistically significant difference between the iNOS immunoreactivity of the interstitial cells in the ovarian parenchyma of the rats in the experimental and control groups (p> 0.05).

Increased intracellular Ca2+ decreases cisplatin resistance by regulating iNOS expression in human ovarian cancer cells.

Previous studies have reported that intracellular Ca2+ signals and inducible nitric oxide synthase (iNOS) are involved in cell apoptosis. However, the role of iNOS in cisplatin resistance in ovarian cancer remains unclear. Here, we demonstrate that SKOV3/DDP ovarian cancer cells were more resistant to cisplatin than were SKOV3 ovarian cancer cells. The expression of intracellular Ca2+ and iNOS was more strongly induced by cisplatin in SKOV3 cells than in SKOV3/DDP cells. TAT-conjugated IP3R-derived peptide (TAT-IDPS) increased cisplatin-induced iNOS expression and apoptosis in SKOV3/DDP cells. 2-Aminoethoxydiphenyl borate (2-APB) decreased cisplatin-induced iNOS expression and apoptosis in SKOV3 cells. Thus, iNOS induction may be a valuable strategy for improving the anti-tumor efficacy of cisplatin in ovarian cancer.

Interleukin-17A mediates cardiomyocyte apoptosis through Stat3-iNOS pathway

Interleukin-17A, a pro-inflammatory cytokine, has a direct proapoptotic effect on cardiomyocytes. However, the specific mechanism has not been clarified. In the present study, an in-vitro model of cardiomyocyte apoptosis induced by IL-17A stimulation was employed and the roles of iNOS and Stat3 involved were investigated. Our data showed that the neonatal mouse cardiomyocytes express IL-17 receptors: IL-17RA and IL-17RC, but did not express IL-17A. Exogenous IL-17A significantly induces iNOS expression and hence the cardiomyocyte apoptosis. Moreover, IL-17A-induced cardiomyocyte apoptosis can be achieved directly via iNOS activation. We further showed that exogenous IL-17A simultaneously triggers Stat3 activation, which in turn inhibits IL-17A-induced iNOS expression in cardiomyocytes. And both ChIP and dual-luciferase results confirmed that Stat3 directly inhibits transcriptional activities of iNOS via binding to its specific promoter region. Consistent with these data, silencing of Stat3 in fact can aggravate IL-17A-triggered cardiomyocyte apoptosis. Finally, using an in vivo myocardial ischemia/reperfusion injury model, we verified that Stat3 inhibition increased iNOS expression and exacerbated cardiomyocyte apoptosis. Thus, our data strongly support the notion that Stat3 plays a compensatory anti-apoptotic role in IL-17A/iNOS-mediated cardiomyocyte apoptosis via inhibiting iNOS transcription, providing a novel molecular mechanism of apoptosis regulation and complicated interactions between IL-17A/iNOS and IL-17A/Stat3 signalings.

MSC signature associated with therapeutical effect in osteoarticular diseases

Adipose derived mesenchymal stromal cells (ASC) are adult stem cells exhibiting functional properties that have open the way for cell-based clinical therapies. Primarily, their capacity of multilineage differentiation has been explored in a number of strategies for skeletal tissue regeneration. More recently, MSCs have been reported to exhibit immunosuppressive as well as healing capacities, to improve angiogenesis and prevent apoptosis or fibrosis through the secretion of paracrine mediators including HGF, IL1RA, IL-6, PDL-1, TSG6, TGF-β1 and PGE2. In normal homeostasis, IL1Ra counterbalances the effect of IL1α and IL1β as a competitive receptor antagonist. MSC immunosuppressive effect is highly variable according to cell population heterogeneity. We compared gene expression of huMSC with high potential to inhibit T cells response with low efficient MSC through gene array. We identified a signature associated with high immunoregulatory function. High expression of ITPR1, NR4A3, ABCA1, LIPG ADAMTS4 as well as expression of CD109, ITGA10, IL6R was associated with increased T cell suppressive effect in vitro. We performed preclinical models of osteoarthritis, and showed that a local injection of ASC showed a reduction of synovitis, reduction of osteophytes, joint stabilization, reducing the score of cartilage lesions. We then investigated the role of iNOS, IL6 and IL1RA respectively using the inflammatory collagen-induced arthritis model. In contrast to wt MSCs, which reduced both the incidence and clinical signs of arthritis, IL1RA–/– MSCs were not able to prevent arthritis progression and even worsened the arthritic symptoms. μCT analysis showed a protection against bone erosion by wt MSCs but not in IL1RA–/–MSCs-treated mice. We found a decrease of the number of CD4 + IFN-γ+ Th1 and CD4 + IL-17+ Th17 lymphocytes in the spleens of wt MSCs-treated mice as compared to IL1RA–/– MSCs-treated mice. This work was completed by toxicology data showing the excellent tolerance of the local injection of ADSC and biodistribution showing the persistence of cells after 6 months in murine models. We conducted a open-label phase 1 trial including 18 patients with severe osteoarthritis of the knee in failure of conventional therapies (62.5% were KL IV) at two sites, Montpellier and Wurzburg. Mean age was 61 years, with a 10 years history of knee OA. The patient received a single injection of autologous ASC 15 days after lipoaspiration (2.106, 107 or 5.107) through intra-articular injection. The primary outcome measure of effectiveness was patient-reported WOMAC pain subscores by VAS in the affected knee at week 12. Secondary outcome measures included Outcome Measures in Rheumatology Clinical Trials and Osteoarthritis Research Society International (OMERACT OARSI) responses. We observed a decrease of the VAS Pain (73 ± 11 mm day 0 to 32 ± 23 month 3), and of WOMAC (50 ± 18 to 25 ± 7 month 3). This study confirms the feasibility and safety of local injection of autologous cells from adipose tissue and suggested that the most effective dose was 107 autologous cells. Moreover, a molecular signature associated with increase immunosuppressive potential is described.

Embryonic stem cell-derived osteocytes are capable of responding to mechanical oscillatory hydrostatic pressure

Osteoblasts can be derived from embryonic stem cells (ESCs) by a 30 day differentiation process, whereupon cells spontaneously differentiate upon removal of LIF and respond to exogenously added 1,25α(OH)2 vitamin D3 with enhanced matrix mineralization. However, bone is a load-bearing tissue that has to perform under dynamic pressure changes during daily movement, a capacity that is executed by osteocytes. At present, it is unclear whether ESC-derived osteogenic cultures contain osteocytes and whether these are capable of responding to a relevant cyclic hydrostatic compression stimulus.Here, we show that ESC-osteoblastogenesis is followed by the generation of osteocytes and then mechanically load ESC-derived osteogenic cultures in a compression chamber using a cyclic loading protocol. Following mechanical loading of the cells, iNOS mRNA was upregulated 31-fold, which was consistent with a role for iNOS as an immediate early mechanoresponsive gene. Further analysis of matrix and bone-specific genes suggested a cellular response in favor of matrix remodeling. Immediate iNOS upregulation also correlated with a concomitant increase in Ctnnb1 and Tcf7l2 mRNAs along with increased nuclear TCF transcriptional activity, while the mRNA for the repressive Tcf7l1 was downregulated, providing a possible mechanistic explanation for the noted matrix remodeling. We conclude that ESC-derived osteocytes are capable of responding to relevant mechanical cues, at least such that mimic oscillatory compression stress, which not only provides new basic understanding, but also information that likely will be important for their use in cell-based regenerative therapies.

Expression of inducible nitric oxide synthase in carcinomas and sarcomas affecting the oral cavity.

Context:Inducible nitric oxide synthase (iNOS) is a cytoplasmic enzyme which plays a crucial role in the pathogenesis of oral carcinomas and sarcomas.Aims:The objective of this study was to analyze the immunohistochemical expression of iNOS in carcinomas and sarcomas affecting the oral cavity in order to understand the possible role of iNOS in their biologic behavior and to correlate iNOS expression with lymph node metastasis in carcinomas and sarcomas.Settings and Design:Patients, who attended the oral diagnosis department of Vinayaka Missions Sankarachariyar Dental College, were screened, for the purpose of the study. Besides these, paraffin-embedded tissue blocks were also retrieved from archives of the Oral and Maxillofacial Pathology Department. A total of 40 cases (20 carcinomas and 20 sarcomas) were selected for the study.Subjects and Methods:A total of 40 cases (20 carcinomas and 20 sarcomas) were selected for the study. Five apparently normal tissues were obtained from the tumor adjacent normal tissue to be used as a control. These were subjected to immunohistochemical staining using antibody to iNOS and evaluated.Statistical Analysis Used:The results were analyzed using the Chi-square test.Results:Among the 20 carcinomas 19 showed a positive immunoreactivity for iNOS and 1 case was negative. Among the 19 immunopositive iNOS cases of carcinomas, 15 cases showed positive lymph node metastasis. Among the sarcomas, positive immunoreactivity for iNOS was seen in 10 hard tissue sarcomas, while the remaining 10 soft tissue sarcomas were negative for iNOS expression. The results were analyzed using the Chi-square test.Conclusions:iNOS is a reliable marker for lymph node metastasis in carcinomas irrespective of the histologic grade. The high expression in carcinomas shows that the carcinomas elaborate more angiogenesis for growth compared with the sarcomas with the exception of hard tissue sarcomas.

Expression of inducible nitric oxide synthase in the epithelial linings of odontogenic keratocyst, dentigerous cyst and radicular cyst: a pathological insight.

Background:The present study is aimed at analyzing the expression of inducible nitric oxide synthase (iNOS) in the epithelial lining of odontogenic keratocyst (OKC), dentigerous cyst (DC), radicular cyst (RC) in order to understand the possible role of iNOS with special reference to its neoplastic nature and local aggressive of cysts.Aim:The primary aim of the following study is to analyze the immunohistochemical expression of iNOS and secondary aim is to compare the iNOS expression, pattern and intensity of staining among the epithelial linings of OKC, DC and RC.Materials and Methods:iNOS in the epithelial lining cells were analyzing in 10 OKC's, 10 DC's and 10 RC's using immunohistochemistry. The percentage of positive cells was assessed and presented as mean ± standard deviation. The correlation with respect to the intensity and percentage of staining within the epithelial linings of OKCs, DCs and RCs was carried out using (analysis of variance and Student's t-test) Chi-square test.Results:Staining intensity of iNOS portion was seen in the entire thickness of the epithelial linings of OKC, whereas in DC's only one case had entire thickness of the epithelial lining staining and in RC's none of the cases showed entire thickness of staining. On comparing the staining intensity of iNOS between OKC, DC and RC groups, using Chi-square test, there was a statistically significant difference between these groups (P < 0.01). On analyzing the immuno-reactivity of iNOS in OKC, DC and RC there was a positive variable expression iNOS between the cysts.Conclusion:iNOS was over expressed in OKCs when compared with DC and RC suggesting that iNOS may contribute to the aggressive behavior of OKC. This is yet another evidence to support that OKC is the neoplasm.