eNOS Expression Research Articles

Changes of rat’s brain vesseles after air shock wave exposure

Mild blast-induced traumatic brain injury is common among the military, resulting in cognitive impairment, reduced socialization, which leads to disability and, as a result, a deterioration in the quality of life. It is considered that blood-brain barrier disruption and microvascular dysfunction are the key to this type of injury. The purpose of study was to study changes in brain vessels after air shock wave exposure. The study was carried out on 48 mature male Wistar rats, which were randomly divided into 2 groups: an experimental group, in which animals were subjected to inhalation anesthesia using halothane and exposed to a shock wave with an overpressure of 26.4±3.6 kPa, and a Sham group. After simulation of injury on days 1st, 3rd, 7th, 14th, and 21st, the rats were euthanized and the brain was removed and after all subjected to standard histological procedures and stained with hematoxylin and eosin. For immunohistochemical studies, as primary antibodies were used eNOS. The finished preparations were examined by light microscopy and photographed. Disorders of the cerebral vessels in experimental rats were detected from day 1st of the posttraumatic period. It was found that the blast wave led to vascular rupture, as well as increased vascular permeability with diapedesis of red blood cells and cerebral edema for up to 21st days. Focal violations of the vascular wall integrity in cortical and hippocampal hemocapillaries, venular link of the submembrane vessels; changes in the morphology of the metabolic vessels endothelium; uneven blood filling of the brain vessels were of major importance. These changes indicate that increased eNOS expression leads to dilation of cerebral vessels, which is a compensatory mechanism in response to injury to improve cerebral blood circulation. However, eNOS is not involved in vasodilation, which we observed up to 21st day post-trauma.

KGRT peptide incorporated hydrogel with antibacterial activity for wound healing by optimizing cellular functions via ERK/eNOS signaling

Bacterial infected wounds, which is characterized by easy infection, multiple inflammation and slow healing, is a complex symptom, resulting from metabolic disorder of the wound microenvironment. In this study, a series of self-healing double-network hydrogels based on KGRT peptide (Lys-Gly-Arg-Thr) with antibacterial, anti-inflammatory and optimizing cellular functions were designed to promote the healing of infected wounds with full-thickness skin defects. Moreover, the dextran hydrogelintroduces a large number of side chains, which are entangled with each other in the Schiff base network to form an interpenetrating structure. The hydrogel might regulate cell metabolism, differentiation and vascular endothelial growth factor (VEGF) function. Importantly, both in vitro and in vivo data showed that hydrogel not only has good antibacterial properties (99.8 %), but also can eradicate bacterial biofilm, effectively reduce inflammation (down-regulated IL-1β, TNF-α and ROS) and accelerate chronic wound healing process by speeding-up wound closure, increasing granulation tissue thickness, collagen deposition, angiogenesis (up-regulated CD31). The hydrogel could up-regulate mRNA expression of PI3K, AKT, ERK, eNOS, HIF-1α and VEGF, which were correlated with wound healing. Consistently, the hydrogel could promote infected wounds healing and inhibit inflammation through ERK/eNOS signaling pathway. Collectively, hydrogel has excellent clinical application potential for promoting infected wound healing.

Effects of heterogeneous surface characteristics on hemocompatibility and cytocompatibility of bacterial nanocellulose

The surface properties of cardiovascular biomaterials play a critical role in their biological responses. Although bacterial nanocellulose (BNC) materials have exhibited potential applications in cardiovascular implants, the impact of their surface characteristics on biocompatibility has rarely been studied. This study investigated the mechanism for the biocompatibility induced by the physicochemical properties of both sides of BNC. With greater wettability and smoothness, the upper BNC surface reduced protein adsorption by 25 % compared with the lower surface. This prolonged the plasma re-calcification time by 14 % in venous blood. Further, compared with the lower BNC surface, the upper BNC surface prolonged the activated partial thromboplastin time by 5 % and 4 % in arterial and venous blood, respectively. Moreover, the lower BNC surface with lesser rigidity, higher roughness, and sparser fiber structure promoted cell adhesion. The lower BNC surface enhanced the proliferation rate of L929 and HUVECs cells by 15 % and 13 %, respectively, compared with the upper BNC surface. With lesser stiffness, the lower BNC surface upregulated the expressions of CD31 and eNOS while down-regulating the ICAM-1 expression - This promoted the proliferation of HUVECs. The findings of this study will provide valuable insights into the design of blood contact materials and cardiovascular implants.

A 2-week treatment with 5-azacytidine improved the hypercontractility state in prostate from obese mice: Role of the nitric oxide-cyclic guanosine monophosphate signalling pathway.

Benign prostatic hyperplasia (BPH) is characterised by increases in prostate volume and contraction. Downregulation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signalling pathway contributes to prostate dysfunctions. Previous studies in cancer cells or vessels have shown that the epigenetic mechanisms control the gene and protein expression of the enzymes involved in the production of NO and cGMP. This study is aimed to evaluate the effect of a 2-week treatment of 5-azacytidine (5-AZA), a DNA-methyltransferase inhibitor, in the prostate function of mice fed with a high-fat diet. Functional, histological, biochemical and molecular assays were carried out. Obese mice presented greater prostate weight, α-actin expression and contractile response induced by the α-1adrenoceptors agonist. The relaxation induced by the NO-donor and the protein expression of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) were significantly decreased in the prostate of obese mice. The treatment with 5-AZA reverted the higher expression of α-actin, reduced the hypercontractility state of the prostate and increased the expression of eNOS and sGC and intraprostatic levels of cGMP. When prostates from obese mice treated with 5-AZA were incubated in vitro with inhibitors of the NOS or sGC, the inhibitory effect of 5-AZA was reverted, therefore, showing the involvement of NO and cGMP. In conclusion, our study paves the way to develop or repurpose therapies that recover the expression of eNOS and sGC and, hence, to improve prostate function in BPH.

Sodium valproate treatment reverses endothelial dysfunction in aorta from rabbits with acute myocardial infarction

Sodium valproate (VPA), a histone deacetylase (HDAC) inhibitor, could be a promising candidate to treat acute myocardial infarction (AMI). In this study, AMI was induced in New Zealand White rabbits by occluding the left circumflex coronary artery for 1 h, followed by reperfusion. The animals were distributed into three experimental groups: the sham-operated group (SHAM), the AMI group and the AMI + VPA group (AMI treated with VPA 500 mg/kg/day). After 5 weeks, abdominal aorta was removed and used for isometric recording of tension in organ baths or protein expression by Western blot, and plasma for the determination of nitrate/nitrite (NOx) levels by colorimetric assay. Our results indicated that AMI induced a reduction of the endothelium-dependent response to acetylcholine without modifying the endothelium-independent response to sodium nitroprusside, leading to endothelial dysfunction. VPA treatment reversed AMI-induced endothelial dysfunction and even increased NO sensitivity in vascular smooth muscle. This response was consistent with an antioxidant effect of VPA, as it was able to reverse the superoxide dismutase 1 (SOD 1) down-regulation induced by AMI. Our experiments also ruled out that the VPA mechanism was related to eNOS, iNOS, sGC and arginase expression or changes in NOx plasma levels. Therefore, we conclude that VPA improves vasodilation by increasing NO bioavailability, likely due to its antioxidant effect. Since endothelial dysfunction was closely related to AMI, VPA treatment could increase aortic blood flow, making it a potential agent in reperfusion therapy that can prevent the vascular damage.

SOX17/ETV2 improves the direct reprogramming of adult fibroblasts to endothelial cells

An autologous source of vascular endothelial cells (ECs) is valuable for vascular regeneration and tissue engineering without the concern of immune rejection. The transcription factor ETS variant 2 (ETV2) has been shown to directly convert patient fibroblasts into vascular EC-like cells. However, reprogramming efficiency is low and there are limitations in EC functions, such as eNOS expression. In this study, we directly reprogram adult human dermal fibroblasts into reprogrammed ECs (rECs) by overexpressing SOX17 in conjunction with ETV2. We find several advantages to rEC generation using this approach, including improved reprogramming efficiency, increased enrichment of EC genes, formation of large blood vessels carrying blood from the host, and, most importantly, expression of eNOS invivo. From these results, we present an improved method to reprogram adult fibroblasts into functional ECs and posit ideas for the future that could potentially further improve the reprogramming process.

Vascular dysfunction programmed in male rats by topiramate during peripubertal period

AimThe present study evaluated whether topiramate (TPM) treatment during the peripubertal period affects vascular parameters of male rats and whether oxidative stress plays a role in these changes. Main methodsRats were treated with TPM (41 mg/kg/day, gavage) or vehicle (CTR group) from the postnatal day (PND) 28 to 50. At PND 51 and 120 the rats were evaluated for: thoracic aorta reactivity to phenylephrine, in the presence (Endo+) or absence of endothelium (Endo-), to acetylcholine and to sodium nitroprusside (SNP), aortic thickness and endothelial nitric oxide synthase (eNOS) expression. In serum were analyzed: the antioxidant capacity by ferric reducing antioxidant power assay; endogenous antioxidant reduced glutathione, and superoxide anion. Results were expressed as mean ± s.e.m., differences when p < 0.05. Statistics: Two-way ANOVA (and Tukey's) or Student t-test. Key findingsAt PND 51, the contraction induced by phenylephrine in Endo+ ring was higher in TPM when compared to CTR. At PND 120, the aortic sensitivity to acetylcholine in TPM rats was reduced in comparison with CTR. The aortic eNOs expression and the aortic thickness were similar between the groups. At PND 51 and 120, TPM group presented a decrease in antioxidants when compared to CTR groups and at PND 120, in TPM group the superoxide anion was increased. SignificanceTaken together, the treatment of rats with TPM during peripubertal period promoted permanent impairment of endothelial function probably mediated by oxidative stress.

Expression of BAX and eNOS in Rabbit Pancreatic Tissues Injured by Hydrocortisone

Background: There have been cases of acute pancreatitis brought on by steroids, but identifying it is challenging and necessitates careful monitoring. However, according to new research, 3–5% of all cases of illness may be caused by drug-induced acute pancreatitis, making it the third most common cause. Objective: Evaluation of the effect of hydrocortisone injections on pancreatic structure immunohistochemically using BAX and eNOS markers. Methods: White New Zealand female rabbits weighing between 1.2 and 1.5 kg were used, and they were given free access to food. The rabbits were split into six groups, with five animals in each group receiving intramuscular hydrocortisone injections for 14 and 21 days, respectively, at a dose of 5 mg and 20 mg/kg for short and long durations, and two control groups. Results: There was an increase in weight in both long-duration groups (GL1 and GL2) after week 2 of injection when compared to both control and short-duration groups. There was a highly statistical difference in the expression of BAX in both short- and long-duration groups compared to the control group, and there was also a decrease in the expression of BAX when duration increased. Similarly, there was a highly statistical difference in the expression of eNOS in both the GS and GL groups when compared to the control group. Conclusions: The pancreas can be injured by high and low doses of hydrocortisone if used for more than 2 weeks.

Quercetin Prevents HSpertension in Dahl Salt-sensitive Rats Fed a High-salt Diet Through Balancing Endothelial Nitric Oxide Synthase and Sirtuin 1.

A high-salt diet is a leading dietary risk factor for elevated blood pressure and cardiovascular disease. Quercetin reportedly exhibits cardioprotective and antihypertensive therapeutic effects. The objective of this study is to examine the effect of quercetin on high-salt dietinduced elevated blood pressure in Dahl salt-sensitive (SS) rats and determine the underlying molecular mechanism. Rats of the Dahl SS and control SS-13 BN strains were separated into five groups, SS-13 BN rats fed a low-salt diet (BL group), SS-13 BN rats fed a high-salt diet (BH group), Dahl SS rats fed a low-salt diet (SL group), Dahl SS rats fed a high-salt diet (SH group), and SH rats treated with quercetin (SHQ group). Blood pressure was checked three weeks into the course of treatment, and biochemical markers in the urine and serum were examined. Additionally, western blot was done to evaluate the sirtuin 1 (SIRT1) and endothelial nitric oxide synthase (eNOS) expression levels. Immunohistochemical analysis was performed to verify SIRT1 levels. We demonstrated that a high-salt diet elevated blood pressure in both SS-13 BN and Dahl SS rats, and quercetin supplementation alleviated the altered blood pressure. Compared with the SH group, quercetin significantly elevated the protein expression of SIRT1 and eNOS. Immunohistochemistry results further confirmed that quercetin could improve the protein expression of SIRT1. Quercetin reduced blood pressure by enhancing the expression of SIRT1 and eNOS in Dahl SS rats fed a high-salt diet.

Differential 5'-tRNA Fragment Expression in Circulating Preeclampsia Syncytiotrophoblast Vesicles Drives Macrophage Inflammation.

The relationship between placental pathology and the maternal syndrome of preeclampsia is incompletely characterized. Mismatch between placental nutrient supply and fetal demands induces stress in the syncytiotrophoblast, the layer of placenta in direct contact with maternal blood. Such stress alters the content and increases the release of syncytiotrophoblast extracellular vesicles (STB-EVs) into the maternal circulation. We have previously shown 5'-tRNA fragments (5'-tRFs) constitute the majority of small RNA in STB-EVs in healthy pregnancy. 5'-tRFs are produced in response to stress. We hypothesized STB-EV 5'-tRF release might change in preeclampsia. We perfused placentas from 8 women with early-onset preeclampsia and 6 controls, comparing small RNA expression in STB-EVs. We used membrane-affinity columns to isolate maternal plasma vesicles and investigate placental 5'-tRFs in vivo. We quantified 5'-tRFs from circulating STB-EVs using a placental alkaline phosphatase immunoassay. 5'-tRFs and scrambled RNA controls were added to monocyte, macrophage and endothelial cells in culture to investigate transcriptional responses. 5'-tRFs constitute the majority of small RNA in STB-EVs from both preeclampsia and normal pregnancies. More than 900 small RNA fragments are differentially expressed in preeclampsia STB-EVs. Preeclampsia-dysregulated 5'-tRFs are detectable in maternal plasma, where we identified a placentally derived load. 5'-tRF-Glu-CTC, the most abundant preeclampsia-upregulated 5'-tRF in perfusion STB-EVs, is also increased in preeclampsia STB-EVs from maternal plasma. 5'-tRF-Glu-CTC induced inflammation in macrophages but not monocytes. The conditioned media from 5'-tRF-Glu-CTC-activated macrophages reduced eNOS (endothelial NO synthase) expression in endothelial cells. Increased release of syncytiotrophoblast-derived vesicle-bound 5'-tRF-Glu-CTC contributes to preeclampsia pathophysiology.

Mechanism Investigation of Tianxiangdan Against Myocardial Ischemia Based on Network Pharmacology, Molecular Docking, and in vitro Experimental

Objectives: Tianxiangdan capsule (TXD) has been clinically used in the treatment of coronary heart disease angina pectoris. This study aimed to evaluate the mechanisms of TXD against myocardial ischemia and provide evidence for its subsequent clinical application. Materials and Methods: Active components and mechanisms of action of TXD against myocardial ischemia were predicted and analyzed by network pharmacology and molecular docking. The chemical constituents in TXD were determined by HPLC-MS. The oxidative damage model was established using H2O2, which caused myocardial ischemia damage. The MTT assay and Hoechst 33342 staining were used to evaluate cell viability, while cleaved caspase-3 immunofluorescence staining was used to determine cell apoptosis. The fluorescent probe method detected ROS and intracellular Ca2+, while spectrophotometry was used to measure SOD, MDA, and NO levels in myocardial cells. Western blotting was used to detect the expression levels of ERɑ, PI3K, p-PI3K, AKT, p-AKT, and eNOS in cells. Results: It was found that TXD plays a protective role in myocardial ischemia through the estrogen pathway. The TXD drug-containing plasma exhibited increased cell survival rates and suppressed MDA levels, elevated SOD and NO levels, and significantly suppressed ROS levels as well as intracellular Ca2+ levels. Moreover, the TXD drug-containing plasma pretreated cells had significantly suppressed PI3K, p-PI3K, AKT, and p-AKT expression levels, as well as elevated ERɑ and eNOS expression levels. Conclusion: TXD activates estrogen receptor ERɑ, enhances NO release of cardiomyocytes through the estrogen signaling pathway, improves intracellular Ca2+ overload, oxidative stress injury, and apoptosis, and plays a protective role in myocardial ischemia injury.

The Impact of LY487379 or CDPPB on eNOS Expression in the Mouse Brain and the Effect of Joint Administration of Compounds with NO• Releasers on MK-801- or Scopolamine-Driven Cognitive Dysfunction in Mice.

The role of endothelial nitric oxide synthase (eNOS) in the regulation of a variety of biological processes is well established, and its dysfunction contributes to brain pathologies, including schizophrenia or Alzheimer's disease (AD). Positive allosteric modulators (PAMs) of metabotropic glutamate (mGlu) receptors were shown to be effective procognitive compounds, but little is known about their impact on eNOS expression and stability. Here, we investigated the influence of the acute and chronic administration of LY487379 or CDPPB (mGlu2 and mGlu5 PAMs), on eNOS expression in the mouse brain and the effect of the joint administration of the ligands with nitric oxide (NO) releasers, spermineNONOate or DETANONOate, in different combinations of doses, on MK-801- or scopolamine-induced amnesia in the novel object recognition (NOR) test. Our results indicate that both compounds provoked eNOS monomer formation, and CDPPB at a dose of 5 mg/kg exaggerated the effect of MK-801 or scopolamine. The coadministration of spermineNONOate or DETANONOate enhanced the antiamnesic effect of CDPPB or LY487379. The best activity was observed for ineffective or moderate dose combinations. The results indicate that treatment with mGluR2 and mGluR5 PAMs may be burdened with the risk of promoting eNOS uncoupling through the induction of dimer dissociation. Administration of the lowest possible doses of the compounds with NO• donors, which themselves have procognitive efficacy, may be proposed for the treatment of schizophrenia or AD.

Human Coronary Artery Endothelial Cell Response to Porphyromonas gingivalis W83 in a Collagen Three-Dimensional Culture Model.

P. gingivalis has been reported to be an endothelial cell inflammatory response inducer that can lead to endothelial dysfunction processes related to atherosclerosis; however, these studies have been carried out in vitro in cell culture models on two-dimensional (2D) plastic surfaces that do not simulate the natural environment where pathology develops. This work aimed to evaluate the pro-inflammatory response of human coronary artery endothelial cells (HCAECs) to P. gingivalis in a 3D cell culture model compared with a 2D cell culture. HCAECs were cultured for 7 days on type I collagen matrices in both cultures and were stimulated at an MOI of 1 or 100 with live P. gingivalis W83 for 24 h. The expression of the genes COX-2, eNOS, and vWF and the levels of the pro-inflammatory cytokines thromboxane A2 (TXA-2) and prostaglandin I2 (PGI2) were evaluated. P. gingivalis W83 in the 2D cell culture increased IL-8 levels at MOI 100 and decreased MCP-1 levels at both MOI 100 and MOI 1. In contrast, the 3D cell culture induced an increased gene expression of COX-2 at both MOIs and reduced MCP-1 levels at MOI 100, whereas the gene expression of eNOS, vWF, and IL-8 and the levels of TXA2 and PGI2 showed no significant changes. These data suggest that in the collagen 3D culture model, P. gingivalis W83 induces a weak endothelial inflammatory response.

Platelet-derived Growth Factor Activates Pericytes in the Microvessels of Chronic Subdural Hematoma Outer Membranes.

Angiogenesis is one of the growth mechanisms of chronic subdural hematoma (CSDH). Pericytes have been implicated in the capillary sprouting during angiogenesis and are involved in brain ischemia and diabetic retinopathy. This study examined the pericyte expressions in CSDH outer membranes obtained during trepanation surgery. Eight samples of CSDH outer membranes and 35 samples of CSDH fluid were included. NG2, N-cadherin, VE-cadherin, Tie-2, endothelial nitric oxide synthase (eNOS), platelet-derived growth factor (PDGF) receptor-β (PDGFR-β), a well-known marker of pericytes, phosphorylated PDGFR-β at Tyr751, and β-actin expressions, were examined using western blot analysis. PDGFR-β, N-cadherin, and Tie-2 expression levels were also examined using immunohistochemistry. The concentrations of PDGF-BB in CSDH fluid samples were measured using enzyme-linked immunosorbent assay kits. NG2, N-cadherin, VE-cadherin, Tie-2, eNOS, PDGFR-β, and eNOS expressions in CSDH outer membranes were confirmed in all cases. Furthermore, phosphorylated PDGFR-β at Tyr751 was also detected. In addition, PDGFR-β, N-cadherin, and Tie-2 expressions were localized to the endothelial cells of the vessels within CSDH outer membranes by immunohistochemistry. The concentration of PDGF-BB in CSDH fluids was significantly higher than that in cerebrospinal fluid. These findings indicate that PDGF activates pericytes in the microvessels of CSDH outer membranes and suggest that pericytes are crucial in CSDH angiogenesis through the PDGF/PDGFR-β signaling pathway.

Long-term iron supplementation combined with vitamin B6 enhances maximal oxygen uptake and promotes skeletal muscle-specific mitochondrial biogenesis in rats.

Iron is an essential micronutrient that plays a crucial role in various biological processes. Previous studies have shown that iron supplementation is related to exercise performance and endurance capacity improvements. However, the underlying mechanisms responsible for these effects are not well understood. Recent studies have suggested the beneficial impact of iron supplementation on mitochondrial function and its ability to rescue mitochondrial function under adverse stress in vitro and rodents. Based on current knowledge, our study aimed to investigate whether the changes in exercise performance resulting from iron supplementation are associated with its effect on mitochondrial function. In this study, we orally administered an iron-based supplement to rats for 30 consecutive days at a dosage of 0.66 mg iron/kg body weight and vitamin B6 at a dosage of 0.46 mg/kg. Our findings reveal that long-term iron supplementation, in combination with vitamin B6, led to less body weight gained and increased VO2 max in rats. Besides, the treatment substantially increased Complex I- and Complex II-driven ATP production in intact mitochondria isolated from gastrocnemius and cerebellum. However, the treatment did not change basal and succinate-induced ROS production in mitochondria from the cerebellum and skeletal muscle. Furthermore, the iron intervention significantly upregulated several skeletal muscle mitochondrial biogenesis and metabolism-related biomarkers, including PGC-1α, SIRT1, NRF-2, SDHA, HSL, MTOR, and LON-P. However, it did not affect the muscular protein expression of SIRT3, FNDC5, LDH, FIS1, MFN1, eNOS, and nNOS. Interestingly, the iron intervention did not exert similar effects on the hippocampus of rats. In conclusion, our study demonstrates that long-term iron supplementation, in combination with vitamin B6, increases VO2 max, possibly through its positive role in regulating skeletal muscle-specific mitochondrial biogenesis and energy production in rats.

Lymphocyte-Specific Protein 1 Regulates Expression and Stability of Endothelial Nitric Oxide Synthase.

Nitric oxide (NO), synthesized by endothelial nitric oxide synthase (eNOS), plays a critical role in blood pressure regulation. Genome-wide association studies have identified genetic susceptibility loci for hypertension in human lymphocyte-specific protein 1 (LSP1) gene. LSP1 is recognized as modulator of leukocyte extravasation, and endothelial permeability, however, the role of LSP1 in regulation of NO signaling within endothelial cells (ECs) remains unknown. The present study investigated the role of LSP1 in the regulation of eNOS expression and activity utilizing human macrovascular ECs in vitro and LSP1 knockout (KO) mice. In ECs, specific CRISPR-Cas9 genomic editing deleted LSP1 and caused downregulation of eNOS expression. LSP1 gain-of-function through adenovirus-mediated gene transfer was associated with enhanced expression of eNOS. Co-immunoprecipitation and confocal fluorescence microscopy revealed that eNOS and LSP1 formed a protein complex under basal conditions in ECs. Furthermore, LSP1 deficiency in mice promoted significant upregulation and instability of eNOS. Utilizing a mass-spectrometry-based bottom-up proteomics approach, we identified novel truncated forms of eNOS in immunoprecipitates from LSP1 KO aortae. Our experimental data suggest an important role of endothelial LSP1 in regulation of eNOS expression and activity within human ECs and murine vascular tissues.

Exploring the impact of naltrexone on the THBS1/eNOS/NO pathway in osteoporotic bile duct-ligated rats

Hepatic osteodystrophy, a prevalent manifestation of metabolic bone disease, can arise in the context of chronic liver disease. The THBS1-eNOS-NO signaling pathway plays a pivotal role in the maturation of osteoclast precursors. This study aimed to investigate the impact of Naltrexone (NTX) on bone loss by examining the THBS1-eNOS-NO signaling pathways in bile duct ligated (BDL) rats. Male Wistar rats were randomly divided into five groups (n = 10 per group): control, sham-operated + normal saline, BDL + normal saline, sham-operated + NTX (10 mg/kg), and BDL + NTX. Parameters related to liver injury were measured at the study's conclusion, and Masson-trichrome staining was employed to evaluate collagen deposition in liver tissue. Bone THBS-1 and endothelial nitric oxide synthase (eNOS) expression levels were measured using real-time PCR, while the level of bone nitric oxide (NO) was assessed through a colorimetric assay. NTX treatment significantly attenuated the BDL-induced increase in circulating levels of liver enzymes and bilirubin. THBS-1 expression levels, elevated after BDL, were significantly suppressed following NTX administration in the BDL + NTX group. Despite no alterations in eNOS expression between groups, the bone NO level, significantly decreased in the BDL group, was significantly reduced by NTX in the BDL + NTX group. This study partly provides insights into the possible molecular mechanisms in BDL-induced osteoporosis and highlights the modulating effect of NTX on these pathways. Further research is needed to establish the impact of NTX on histomorphometric indexes.

Cardiac endothelial ischemia/reperfusion injury-derived protein damage-associated molecular patterns disrupt the integrity of the endothelial barrier

Human cardiac microvascular endothelial cells (HCMECs) are sensitive to ischemia and vulnerable to damage during reperfusion. The release of damage-associated molecular patterns (DAMPs) during reperfusion induces additional tissue damage. The current study aimed to identify early protein DAMPs in human cardiac microvascular endothelial cells subjected to ischemia-reperfusion injury (IRI) using a proteomic approach and their effect on endothelial cell injury. HCMECs were subjected to 60 min of simulated ischemia and 6 h of reperfusion, which can cause lethal damage. DAMPs in the culture media were subjected to liquid chromatography-tandem mass spectrometry proteomic analysis. The cells were treated with endothelial IRI-derived DAMP medium for 24 h. Endothelial injury was assessed by measuring lactate dehydrogenase activity, morphological features, and the expression of endothelial cadherin, nitric oxide synthase (eNOS), and caveolin-1. The top two upregulated proteins, DNAJ homolog subfamily B member 11 and pyrroline-5-carboxylate reductase 2, are promising and sensitive predictors of cardiac microvascular endothelial damage. HCMECs expose to endothelial IRI-derived DAMP, the lactate dehydrogenase activity was significantly increased compared with the control group (10.15 ± 1.03 vs 17.67 ± 1.19, respectively). Following treatment with endothelial IRI-derived DAMPs, actin-filament dysregulation, and downregulation of vascular endothelial cadherin, caveolin-1, and eNOS expressions were observed, along with cell death. In conclusion, the early protein DAMPs released during cardiac microvascular endothelial IRI could serve as novel candidate biomarkers for acute myocardial IRI. Distinct features of impaired plasma membrane integrity can help identify therapeutic targets to mitigate the detrimental consequences mediated of endothelial IRI-derived DAMPs.

Protective role of protease-activated receptor-2 in anaphylaxis model mice.

Anaphylaxis is a severe life-threatening hypersensitivity reaction induced by mast cell degranulation. Among the various mediators of mast cells, little is known about the role of tryptase. Therefore, we aimed to elucidate the role of protease-activating receptor-2 (PAR-2), a receptor activated by tryptase, in murine anaphylactic models using PAR-2-deficient mice and newly generated tryptase-deficient mice. Anaphylaxis was induced by IgE-dependent and IgE-independent mast cell degranulation in mice. PAR-2 deficiency exacerbated the decrease in body temperature and hypotension during anaphylaxis; however, the number of skin mast cells, degree of mast cell degranulation, and systemic and local vascular hyperpermeability were comparable in PAR-2 knockout and wild-type mice. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), is an indispensable vasodilator in anaphylaxis. In the lungs of anaphylactic mice, PAR-2 deficiency promoted eNOS expression and phosphorylation, suggesting a protective effect of PAR-2 against anaphylaxis by downregulating eNOS activation and expression. Based on the hypothesis that the ligand for PAR-2 in anaphylaxis is mast cell tryptase, tryptase-deficient mice were generated using CRISPR-Cas9. In wild-type mice, the PAR-2 antagonist exacerbated the body temperature drop due to anaphylaxis; however, the effect of the PAR-2 antagonist was abolished in tryptase-deficient mice. These results suggest that tryptase is a possible ligand of PAR-2 in anaphylaxis and that the tryptase/PAR-2 pathway attenuates the anaphylactic response in mice.

The Importance of Nitric Oxide and Oxidative Stress in Atrial High-Rate Episodes in Patients with Cardiac Devices.

Atrial High Rate Episodes (AHRE) are subclinical atrial tachyarrhythmias detectable by cardiac implantable electronic devices (CIEDs). AHREs have been associated with an increased risk of developing atrial fibrillation (AF), thromboembolism, cardiovascular and cerebrovascular events, and mortality. Although recent studies have assessed the value of oxidative stress markers in patients with AF, the relationships between AHRE and oxidative stress markers, including nitric oxide, has not yet been elucidated. This study aims to investigate the relationship between these markers and AHRE. This prospective, cross-sectional study comprised 180 patients with CIEDs. The study population was divided into two groups based on the presence (n = 78) and absense (n = 102) of AHRE to analyze its association with biomarkers. The AHRE (+) group was significantly older, had a higher prevalence of hypertension, higher NT-proBNP (508.8 ± 249 pg/mL vs. 415.3 ± 292.1; P = 0.037), MDA levels (20.9 ± 4.1 μmol/L vs. 19.1 ± 3.1 μmol/L; P = 0.006), and iNOS activity (1,935.9 ± 326.1 pg/mL vs. 1,677.4 ± 363.2 pg/mL; P < 0.001). Logistic regression analysis identified age, hypertension, MDA (odds ratio [OR]: 1.131, 95%CI: 1.009 - 1.268, P = 0.035), inducible nitric oxide synthase (iNOS) activity (OR = 1.002, 95% CI = 1.001 - 1.003, P < 0.001), and endothelial nitric oxide synthase (eNOS) activity (OR = 0.990, 95% CI = 0.986 - 0.984, P < 0.001) as independent predictors of AHRE. The study findings indicated that plasma levels of NT-proBNP, MDA, nitric oxide, and the expression of iNOS and eNOS were significantly associated with AHRE. Moreover, elevated plasma MDA concentrations, increased iNOS activity, and decreased eNOS activity were identified as independent predictors of AHRE.