Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway.

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Nitric oxide controls proliferation of Leishmania major by inhibiting the recruitment of permissive host cells.

Nitric oxide (NO) is an important antimicrobial effector but also prevents unnecessary tissue damage by shutting down the recruitment of monocyte-derived phagocytes. Intracellular pathogens such as Leishmania major can hijack these cells as a niche for replication. Thus, NO might exert containment by restricting the availability of the cellular niche required for efficient pathogen proliferation. However, such indirect modes of action remain to be established. By combining mathematical modeling with intravital 2-photon biosensors of pathogen viability and proliferation, we show that low L. major proliferation results not from direct NO impact on the pathogen but from reduced availability of proliferation-permissive host cells. Although inhibiting NO production increases recruitment of these cells, and thus pathogen proliferation, blocking cell recruitment uncouples the NO effect from pathogen proliferation. Therefore, NO fulfills two distinct functions for L. major containment: permitting direct killing and restricting the supply of proliferation-permissive host cells.

IL-10-Dependent Crosstalk between Murine Marginal Zone B Cells, Macrophages, and CD8α+ Dendritic Cells Promotes Listeria monocytogenes Infection.

Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.

Stress-induced pseudokinase TRB3 augments IL1ß signaling by interacting with Flightless homolog 1.

Interleukin-1ß is one of the most potent inducers of beta cell inflammation in the lead-up to type 1 diabetes. We have previously reported that IL1ß-stimulated pancreatic islets from mice with genetic ablation of stress-induced pseudokinase TRB3(TRB3KO) show attenuated activation kinetics for the MAP3K MLK3 and JNK stress kinases. However, JNK signaling constitutes only a portion of the cytokine-induced inflammatory response. Here we report that TRB3KO islets also show a decrease in amplitude and duration of IL1ß-induced phosphorylation of TAK1 and IKK, kinases that drive the potent NF-κB proinflammatory signaling pathway. We observed that TRB3KO islets display decreased cytokine-induced beta cell death, preceded by a decrease in select downstream NF-κB targets, including iNOS/NOS2 (inducible nitric oxide synthase), a mediator of beta cell dysfunction and death. Thus, loss of TRB3 attenuates both pathways required for a cytokine-inducible, proapoptotic response in beta cells. In order to better understand the molecular basis of TRB3-enhanced, post-receptor IL1ß signaling, we interrogated the TRB3 interactome using coimmunoprecipitation followed by mass spectrometry to identify immunomodulatory protein Flightless homolog 1 (Fli1) as a novel, TRB3-interacting protein. We show that TRB3 binds and disrupts Fli1-dependent sequestration of MyD88, thereby increasing availability of this most proximal adaptor required for IL1ß receptor-dependent signaling. Fli1 sequesters MyD88 in a multiprotein complex resulting in a brake on the assembly of downstream signaling complexes. By interacting with Fli1, we propose that TRB3 lifts the brake on IL1ß signaling to augment the proinflammatory response in beta cells.

Amelioration of endothelial integrity by 3,5,4'-trihydroxy-trans-stilbene against high-fat-diet-induced obesity and -associated vasculopathy and myocardial infarction in rats, targeting TLR4/MyD88/NF-κB/iNOS signaling cascade.

Exacerbated expression of TLR4 protein (foremost pattern recognition receptor) during obesity could trigger NF-κB/iNOS signaling through linker protein (MyD88), predisposed to an indispensable inflammatory response. The induction of this detrimental cascade leads to myocardial and vascular abnormalities. Molecular docking was studied for protein-ligand interaction between these potential targets and resveratrol. The pre-treatment of resveratrol (20 mg/kg/p.o/per day for ten weeks) was given to investigate the therapeutic effect against HFD-induced obesity and associated vascular endothelial dysfunction (VED) and myocardial infarction (MI) in Wistar rats. In addition to accessing the levels of serum biomarkers for VED and MI, oxidative stress, inflammatory cytokines, and histopathology of these tissues were investigated. Lipopolysaccharide (for receptor activation) and protein expression analysis were introduced to explore the mechanistic involvement of TLR4/MyD88/NF-κB/iNOS signaling. Assessment of in-silico analysis showed significant interaction between protein and ligand. The involvement of this proposed signaling (TLR4/MyD88/NF-κB/iNOS) was further endorsed by the impact of lipopolysaccharide and protein expression analysis in obese and treated rats. Moreover, resveratrol pre-treated rats showed significantly lowered cardio and vascular damage measured by the distinct down expression of the TLR4/MyD88/NF-κB/iNOS pathway by resveratrol treatment endorses its ameliorative effect against VED and MI.

Elevated SLC7A2 expression is associated with an abnormal neuroinflammatory response and nitrosative stress in Huntington's disease.

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for L-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington's disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR-Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients.

4R-cembranoid suppresses glial cells inflammatory phenotypes and prevents hippocampal neuronal loss in LPS-treated mice.

Chronic neuroinflammation has been implicated in neurodegenerative disease pathogenesis. A key feature of neuroinflammation is neuronal loss and glial activation, including microglia and astrocytes. 4R-cembranoid (4R) is a natural compound that inhibits hippocampal pro-inflammatory cytokines and increases memory function in mice. We used the lipopolysaccharide (LPS) injection model to study the effect of 4R on neuronal density and microglia and astrocyte activation. C57BL/6J wild-type mice were injected with LPS (5 mg/kg) and 2 h later received either 4R (6 mg/kg) or vehicle. Mice were sacrificed after 72 h for analysis of brain pathology. Confocal images of brain sections immunostained for microglial, astrocyte, and neuronal markers were used to quantify cellular hippocampal phenotypes and neurons. Hippocampal lysates were used to measure the expression levels of neuronal nuclear protein (NeuN), inducible nitrous oxide synthase (iNOS), arginase-1, thrombospondin-1 (THBS1), glial cell-derived neurotrophic factor (GDNF), and orosomucoid-2 (ORM2) by western blot. iNOS and arginase-1 are widely used protein markers of pro- and anti-inflammatory microglia, respectively. GDNF promotes neuronal survival, and ORM2 and THBS1 are astrocytic proteins that regulate synaptic plasticity and inhibit microglial activation. 4R administration significantly reduced neuronal loss and the number of pro-inflammatory microglia 72 h after LPS injection. It also decreased the expression of the pro-inflammatory protein iNOS while increasing arginase-1 expression, supporting its anti-inflammatory role. The protein expression of THBS1, GDNF, and ORM2 was increased by 4R. Our data show that 4R preserves the integrity of hippocampal neurons against LPS-induced neuroinflammation in mice.

Fish arginase constrains excessive production of nitric oxide and limits mitochondrial damage during Aeromonas hydrophila infection.

Bacteria-enhanced inducible nitric oxide synthase (iNOS) overproduces nitric oxide (NO) leading to mitochondrial and cellular damage. In mammals, arginase (ARG), the enzyme consuming the same substrate l-arginine with iNOS, was believed to inhibit iNOS activity by competing the substrate. But in fish, this conception has been widely challenged. In this study, the gene expression using real-time quantitative PCR (RT-qPCR) technology showed that when stimulated by Aeromonas hydrophila (A. hydrophila), grass carp (gc) iNOS was up-regulated in head kidney monocytes/macrophages (M0/MФ), and its changes were not detected in the whole tissue of liver or spleen, showing a high degree of cell-specific expression pattern. At the same time, gcARG2 had a high basal expression in tissues and was up-regulated by A. hydrophila stimulation. Next, phthalaldehyde-primaquine reaction was first used in the determination of intracellular urea in fish cells. It was found that the induced gcARG2 led to an increase in the intracellular urea content. Moreover, urea and NO production in M0/MФ were increased in a substrate dose-dependent manner from 30 to 100 µM of l-arginine and reached the highest yield at 300 and 3000 µM of l-arginine, respectively. Furthermore, head kidney M0/MФ was cultured in RPMI1640 medium containing physiological concentration (500 µM) of l-arginine to evaluate the effect of ARG. Under A. hydrophila stimulation, treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) showed that inhibition of arginase could further enhance the NO production stimulated by A. hydrophila. This in turn led to a cumulation in peroxynitrite (ONOO-) content and an injury of the mitochondrial membrane potential. Our study showed for the first time that fish ARG in head kidney M0/MФ can limit excessive production of NO and harmful products by iNOS to maintain mitochondrial and cellular homeostasis.

Development of novel nitric oxide production inhibitors based on the 7H-pyrrolo[2,3-d]pyrimidine scaffold.

Nitric oxide (NO), the smallest signaling molecule known, can be excessively produced by overexpressed inducible nitric oxide synthase (iNOS), and eventually leads to multiple inflammatory related diseases. Thus, reducing the overexpression of NO represents as very potential anti-inflammatory strategy. In current study, a series of compounds were designed and synthesized based on the hybridization of 7H-pyrrolo[2,3-d]pyrimidine and cinnamamide fragments in order to develop novel NO production inhibitors. Among them, compound S2h displayed a vigorous inhibitory activity on NO production with an IC50 value of 3.21 ± 0.67 µM, which was much lower than that of the positive control Nω-nitro-L-arginine (L-NNA, IC50 = 28.36 ± 3.13 µM). Due to its obeying Lipinski's and Veber's rules that guarantee compounds with good oral bioavailability, S2h effectively suppressed the paw swelling in carrageenan-induced mice. Additionally, compound S2h formed clear interactions with iNOS protein according to the docking analysis. Therefore, compounds S2h is a promising lead compound for further development of potent iNOS inhibitors or anti-inflammatory agents.

Effect of peripheral blood mononuclear cells on ischemia-reperfusion injury of sciatic nerve of adult male albino rat: histological, immunohistochemical, and ultrastructural study.

Ischemia/reperfusion (I/R) injury of sciatic nerve is a serious condition that results in nerve fiber degeneration, and reperfusion causes oxidative injury. Peripheral blood mononuclear cells (PBMNCs) have neuroregenerative power. This study was carried out to evaluate the potential ameliorative effect of PBMNCs on changes induced by I/R injury of the sciatic nerve. Fifty adult male albino rats were divided into donor and experimental groups that were subdivided into four groups: group I (control group), group II received 50 µL PBNMCs once intravenously via the tail vein, group III rubber tourniquet was placed around their Rt hind limb root for 2 hours to cause ischemia, group IV was subjected to limb ischemia as group III, then they were injected with 50 ul PBMNCs as group II before reperfusion. I/R injury showed disorganization of nerve fascicles with wide spaces in between nerve fibers. The mean area of collagen fibers, iNOS immunoexpression, and number of GFAP-positive Schwann cells of myelinated fibers showed a highly significant increase, while a highly significant reduction in the G-ratio and neurofilament immunoexpression was observed. Myelin splitting, invagination, evagination, and myelin figures were detected. PBMNC-treated group showed a marked improvement that was confirmed by histological, immunohistochemical, and ultrastructural findings.

Oxidative Stress in Sepsis: A Focus on Cardiac Pathology.

This study aims to analyze post-mortem human cardiac specimens, to verify and evaluate the existence or extent of oxidative stress in subjects whose cause of death has been traced to sepsis, through immunohistological oxidative/nitrosative stress markers. Indeed, in the present study, i-NOS, NOX2, and nitrotyrosine markers were higher expressed in the septic death group when compared to the control group, associated with also a significant increase in 8-OHdG, highlighting the pivotal role of oxidative stress in septic etiopathogenesis. In particular, 70% of cardiomyocyte nuclei from septic death specimens showed positivity for 8-OHdG. Furthermore, intense and massive NOX2-positive myocyte immunoreaction was noticed in the septic group, as nitrotyrosine immunostaining intense reaction was found in the cardiac cells. These results demonstrated a correlation between oxidative and nitrosative stress imbalance and the pathophysiology of cardiac dysfunction documented in cases of sepsis. Therefore, subsequent studies will focus on the expression of oxidative stress markers in other organs and tissues, as well as on the involvement of the intracellular pattern of apoptosis, to better clarify the complex pathogenesis of multi-organ failure, leading to support the rationale for including therapies targeting redox abnormalities in the management of septic patients.

Inducible nitric oxide synthase activity mediates TNF-α-induced endothelial cell dysfunction.

Inducible nitric oxide synthase (iNOS) and vascular endothelial dysfunction have been implicated in the development and progression of atherosclerosis. This study aimed to elucidate the role of iNOS in vascular endothelial dysfunction. Ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrometry combined with multivariate data analysis was used to characterize the metabolic changes in human umbilical vein endothelial cells (HUVECs) in response to different treatment conditions. In addition, molecular biology techniques were employed to explain the molecular mechanisms underlying the role of iNOS in vascular endothelial dysfunction. Tumor necrosis factor-α (TNF-α) enhances the expression of iNOS, TXNIP, and the level of reactive oxygen species (ROS) facilitates the entry of nuclear factor-κB (NF-κB) into the nucleus and promotes injury in HUVECs. iNOS deficiency reversed the TNF-α-mediated pathological changes in HUVECs. Moreover, TNF-α increased the expression of tumor necrosis factor receptor-2 (TNFR-2) and the levels of p-IκBα and IL-6 proteins and CD31, ICAM-1, and VCAM-1 protein expression, which was significantly reduced in HUVECs with iNOS deficiency. In addition, treating HUVECs in the absence or presence of TNF-α or iNOS, respectively, enabled the identification of putative endogenous biomarkers associated with endothelial dysfunction. These biomarkers were involved in critical metabolic pathways, including glycosylphosphatidylinositol-anchor biosynthesis, amino acid metabolism, sphingolipid metabolism, and fatty acid metabolism. iNOS deficiency during vascular endothelial dysfunction may affect the expression of TNFR-2, vascular adhesion factors, and the level of ROS via cellular metabolic changes, thereby attenuating vascular endothelial dysfunction.NEW & NOTEWORTHY Inducible nitric oxide synthase (iNOS) deficiency during vascular endothelial dysfunction may affect the expression of tumor necrosis factor receptor-2 and vascular adhesion factors via cellular metabolic changes, thereby attenuating vascular endothelial dysfunction.

Arginase 1 expression is increased during hepatic stellate cell activation and facilitates collagen synthesis.

Activation of hepatic stellate cells (HSC) is a key event in the initiation of liver fibrosis. Activated HSCs proliferate and secrete excessive amounts of extracellular matrix (ECM), disturbing liver architecture and function, leading to fibrosis and eventually cirrhosis. Collagen is the most abundant constituent of ECM and proline is the most abundant amino acid of collagen. Arginine is the precursor in the biosynthetic pathway of proline. Arginine is the exclusive substrate of both nitric oxide synthase (NOS) and arginase. NOS is an M1 (proinflammatory) marker of macrophage polarization whereas arginase-1 (Arg1) is an M2 (profibrogenic) marker of macrophage polarization. Differential expression of NOS and Arg1 has not been studied in HSCs yet. To identify the expression profile of arginine catabolic enzymes during HSC activation and to investigate their role in HSC activation, primary rat HSCs were cultured-activated for 7 days and expression of iNOS and Arg1 were investigated. Nor-NOHA was used as a specific and reversible arginase inhibitor. During HSC activation, iNOS expression decreased whereas Arg1 expression increased. Inhibition of Arg1 in activated HSCs efficiently inhibited collagen production but not cell proliferation. HSC activation is accompanied by a switch of arginine catabolism from iNOS to Arg1. Inhibition of Arg1 decreases collagen synthesis. Therefore, we conclude that Arg1 can be a therapeutic target for the inhibition of liver fibrogenesis.

Paneth cell-derived iNOS is required to maintain homeostasis in the intestinal stem cell niche.

BACKGROUND: Mammalian intestinal epithelium constantly undergoes rapid self-renewal and regeneration sustained by intestinal stem cells (ISCs) within crypts. Inducible nitric oxide synthase (iNOS) is an important regulator in tissue homeostasis and inflammation. However, the functions of iNOS on ISCs have not been clarified. Here, we aimed to investigate the expression pattern of inducible nitric oxide synthase (iNOS) within crypts and explore its function in the homeostatic maintenance of the ISC niche. METHODS: Expression of iNOS was determined by tissue staining and qPCR. iNOS-/- and Lgr5 transgenic mice were used to explore the influence of iNOS ablation on ISC proliferation and differentiation. Enteroids were cultured to study the effect of iNOS on ISCs in vitro. Ileum samples from wild-type and iNOS-/- mice were collected for RNA-Seq to explore the molecular mechanisms by which iNOS regulates ISCs. RESULTS: iNOS was physiologically expressed in Paneth cells. Knockout of iNOS led to apparent morphological changes in the intestine, including a decrease in the small intestine length and in the heights of both villi and crypts. Knockout of iNOS decreased the number of Ki67+ or BrdU+ proliferative cells in crypts. Loss of iNOS increased the number of Olfm4+ ISCs but inhibited the differentiation and migration of Lgr5+ ISCs in vivo. iNOS depletion also inhibited enteroid formation and the budding efficiency of crypts in vitro. Moreover, iNOS deficiency altered gluconeogenesis and the adaptive immune response in the ileum transcriptome. CONCLUSION: Paneth cell-derived iNOS is required to maintain a healthy ISC niche, and Knockout of iNOS hinders ISC function in mice. Therefore, iNOS represents a potential target for the development of new drugs and other therapeutic interventions for intestinal disorders.

Effect of nitric oxide synthase gene polymorphism on inflammatory response in patients with chronic obstructive pulmonary disease.

This study aimed to investigate the association between nitric oxide synthase gene polymorphisms and the inflammatory responses in patients with 'fast-' and 'slow-' developing chronic obstructive pulmonary disease (COPD). In the main process, 190 patients with slow-developing COPD, 94 patients with fast-developing COPD and 105 healthy volunteers were selected for inclusion. Endothelial nitric oxide synthase (eNOS) was detected using western-blot eNOS sites, and inducible nitric oxide synthase (iNOS) was detected through SNPshot. T helper 17 cells (Th17) and regulator T (Treg) cells were detected via flow cytometry, and interferon-gamma, tumour necrosis factor-alpha, interleukin (IL)-17, IL-10, IL-6, IL-4 and IL-2 were detected using a cytometric bead array. The final results and conclusions drawn from the tests suggest that Th17/Treg-mediated immune inflammation plays an important role in the pathogenesis of COPD, but whether it affects the development of COPD needs further investigation. Overall, COPD patients with a young age of onset, young age of smoking initiation and small body mass index, as well as COPD patients with CC at rs3729508 in the iNOS gene and non-GG at rs7830 in the eNOS gene, may be more likely to contract fast-developing COPD.

Hypoglycaemia aggravates impaired endothelial-dependent vasodilation in diabetes by suppressing endothelial nitric oxide synthase activity and stimulating inducible nitric oxide synthase expression.

BACKGROUND: Diabetes exacerbates vascular injury by triggering endothelial dysfunction. Endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) both play major roles in endothelial dysfunction. However, effects of hypoglycaemia, the main complication of the insulin therapy to the glycemic control in diabetes, on eNOS activity and iNOS expression, and underlying mechanisms in diabetes remain unknown. Hence, we aimed to determine the effects of hypoglycaemia on eNOS activity and iNOS expression in different arterial beds of diabetic rats. METHODS: Sprague-Dawley rats were subjected to Streptozotocin (STZ) combined with high fat diet (HFD) to induce diabetes and then received insulin injection to attain acute and recurrent hypoglycaemia. Immunoblotting was used to analyse the phosphorylation and O-glycosylation status of eNOS and iNOS level from thoracic aorta and mesenteric artery tissue. Indicators of oxidative stress from plasm were determined, and endothelial-dependent vasodilation was detected via wire myograph system. RESULTS: Hypoglycaemia was associated with a marked increase in eNOS O-GlcNAcylation and decrease in Serine (Ser)-1177 phosphorylation from thoracic aortas and mesenteric arteries. Moreover, hypoglycaemia resulted in elevated phosphorylation of eNOS at Threonine (Thr)-495 site in mesenteric arteries. Besides, changes in these post-translational modifications were associated with increased O-GlcNAc transferase (OGT), decreased phosphorylation of Akt at Ser-473, and increased protein kinase C α subunit (PKCα). iNOS expression was induced in hypoglycaemia. Furthermore, endothelial-dependent vasodilation was impaired under insulin-induced hypoglycaemia, and further in recurrent hypoglycaemia. CONCLUSIONS: Conclusively, these findings strongly indicate that hypoglycaemia-dependent vascular dysfunction in diabetes is mediated through altered eNOS activity and iNOS expression. Therefore, this implies that therapeutic modulation of eNOS activity and iNOS expression in diabetics under intensive glucose control may prevent and treat adverse cardiovascular events.

Sepsis modulates aortic AT1 and P2Y6 receptors to produce vascular hyporeactivity in mice.

PURPOSE: Hyporeactivity to vasopressors leading to multiple organ failure is a serious clinical implication in sepsis. Though the regulatory role of purinoceptors in inflammation is reported, their involvement in sepsis-induced vasoplegia is still unknown. Thus we investigated the effect of sepsis on vascular AT1 and P2Y6 receptors. MATERIALS AND METHODS: Polymicrobial sepsis was induced by cecal ligation and puncture in mice. Vascular reactivity was assessed by organ bath study and aortic mRNA expression of AT1 and P2Y6 was quantified by qRT-PCR. RESULTS: Both angiotensin-II and UDP produced higher contractions in the absence of endothelium as well as following inhibition of nitric oxide synthase. Angiotensin-II mediated aortic contraction was antagonized by losartan (AT1 antagonist), but not by PD123319 (AT2 antagonist) whereas UDP-induced aortic contraction was significantly inhibited by MRS2578 (P2Y6 antagonist). In addition, MRS2578 significantly inhibited the contractile response of Ang-II. Compared to SO mice, angiotensin-II and UDP-induced maximum contraction were found to be significantly attenuated in sepsis. Accordingly, aortic mRNA expression of AT1a receptors was significantly down-regulated while that of P2Y6 receptors was significantly increased in sepsis. 1400 W (a selective iNOS inhibitor) significantly reversed angiotensin-II-induced vascular hyporeactivity in sepsis without affecting UDP-induced hypo-reactivity. CONCLUSION: Sepsis-induced vascular hyporeactivity to angiotensin-II is mediated by enhanced expression of iNOS. Moreover, AT1R-P2Y6 cross talk/heterodimerization could be a novel target for regulating vascular dysfunction in sepsis.

TNF/iNOS/NO pathway mediates host susceptibility to endothelial-dependent circulatory failure and death induced by betacoronavirus infection.

Poor disease outcomes and lethality are directly related to endothelial dysfunction in betacoronavirus infections. Here, we investigated the mechanisms underlying the vascular dysfunction caused by the betacoronaviruses MHV-3 and SARS-CoV-2. Wild-type C57BL/6 (WT) and knockout mice for inducible nitric oxide synthase (iNOS-/-) or TNF receptor 1 (TNFR1-/-) were infected with MHV-3, and K18-hACE2 transgenic mice expressing human ACE2 were infected with SARS-CoV-2. Isometric tension was used to evaluate vascular function. Protein expression was determined by immunofluorescence. Tail-cuff plethysmography and Doppler were used to assess blood pressure and flow, respectively. Nitric oxide (NO) was quantified with the DAF probe. ELISA was used to assess cytokine production. Survival curves were estimated using Kaplan-Meier. MHV-3 infection reduced aortic and vena cava contractility, arterial blood pressure, and blood flow, resulting in death. Resistance mesenteric arteries showed increased contractility. The contractility of the aorta was normalized by removing the endothelium, inhibiting iNOS, genetically deleting iNOS, or scavenging NO. In the aorta, iNOS and phospho-NF-kB p65 subunit expression was enhanced, along with basal NO production. TNF production was increased in plasma and vascular tissue. Genetic deletion of TNFR1 prevented vascular changes triggered by MHV-3, and death. Basal NO production and iNOS expression were also increased by SARS-CoV-2. In conclusion, betacoronavirus induces an endothelium-dependent decrease in contractility in macro-arteries and veins, leading to circulatory failure and death via TNF/iNOS/NO. These data highlight the key role of the vascular endothelium and TNF in the pathogenesis and lethality of coronaviruses.

Nitric oxide synthase inhibitors as potential therapeutic agents for gliomas: A systematic review.

INTRODUCTION: Gliomas represent the most prevalent form of brain tumors, among which glioblastomas are the most malignant subtype. Despite advances in comprehending their biology and treatment strategies, median survival remains disappointingly low. Inflammatory processes involving nitric oxide (NO), critically contribute to glioma formation. The inducible isoform of NO synthase (iNOS) is highly overexpressed in gliomas and has been linked to resistance against temozolomide (TMZ) treatment, neoplastic transformation, and modulation of immune response. While both in vitro and in vivo studies showed the potential of iNOS inhibitors as effective treatments for gliomas, no clinical trials on gliomas have been published. This review aims to summarize the available evidence regarding iNOS as a target for glioma treatment, focusing on clinically relevant data. METHODS: Following PRISMA guidelines, we conducted a systematic review by searching PubMed/Medline, and Embase databases in May 2023. We included studies that investigated the impact of NOS inhibitors on glioma cells using L-NMMA, CM544, PBN, 1400W or l-NAME either alone or combined with TMZ. We extracted data on the NOS inhibitor used, subtype, study setting, animal model or cell lines employed, obtained results, and safety profile. Our inclusion criteria encompassed original articles in English or Spanish, studies with an untreated control group, and a primary outcome focused on the biological effects on glioma cells. RESULTS: Out of 871 articles screened from the aforementioned databases, 37 reports were assessed for eligibility. After excluding studies that did not utilize glioma cells or address the designated outcome, 11 original articles satisfied the inclusion and exclusion criteria. Although no NOS inhibitor has been tested in a published clinical trial, three inhibitors have been evaluated using in vivo models of intracranial gliomas. l-NAME, 1400W, and CM544 were tested in vitro. Co-administration of l-NAME, or CM544 with TMZ showed superior results in vitro compared to individual agent testing. CONCLUSION: Glioblastomas remain a challenging therapeutic target. iNOS inhibitors exhibit substantial potential as treatment options for oncologic lesions, and they have demonstrated a safe toxicity profile in humans for other pathological conditions. Research endeavors should be focused on investigating their potential effects on brain tumors.

Design, Synthesis, anti-inflammatory activity Evaluation, preliminary exploration of the Mechanism, molecule Docking, and structure-activity relationship analysis of batatasin III analogs.

Most clinical drugs used to treat inflammation have serious gastrointestinal, renal, and cardiovascular side effects during long-term treatment. The development of new anti-inflammatory agents from natural products and their derivatives is a powerful approach to overcome these adverse effects. Batatasin III, a bibenzyl natural product, has been found to have anti-inflammatory activity. Compared with other anti-inflammatory agents, batatasin III has a simple and unique structure. Therefore, batatasin III and its analogs might have the potential to treat inflammation with only mild adverse effects as a new type of anti-inflammatory agent. Herein, we synthesized 26 batatasin III analogs and evaluated the anti-inflammatory activity in vitro. Analog 21 significantly inhibited (p < 0.01) nitric oxide production with an IC50 value of 12.95 µM. Western blot analysis further revealed that 21 reduced iNOS, phosphorylated p65, and ß-catenin expression in a concentration-dependent manner. These results indicated that 21 could be a potential lead compound for developing a drug candidate for ulcerative colitis. Molecular docking analysis showed that p65 might be a potential target of 21 for the treatment of inflammatory disease. In addition, we analyzed the structure-activity relationship of the analogs, which provides a basis for future structural modifications.