Phosphoinositide-3-Kinase/Akt-Endothelial Nitric Oxide Synthase Signaling Pathway Mediates the Neuroprotective Effect of Sevoflurane Postconditioning in a Rat Model of Hemorrhagic Shock and Resuscitation.

BACKGROUND: Although extensive reports have demonstrated the neuroprotection of sevoflurane postconditioning in cases of focal and global cerebral ischemia/reperfusion, the underlying mechanisms are not completely elucidated. This study investigated whether this effect is related to endothelial nitric oxide synthase (eNOS) and mediated by the phosphoinositide-3-kinase pathway in a rat model of hemorrhagic shock and resuscitation. METHODS: Adult male Sprague Dawley rats were subjected to hemorrhagic shock for 60 minutes and then resuscitation for 30 minutes in experimental groups. Sevoflurane postconditioning was performed at the beginning of resuscitation to completion. At 24 hours after resuscitation, the brain infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining. The neuronal morphological changes and apoptosis were determined by hematoxylin and eosin staining and immunohistochemistry analysis, respectively. The activity of phosphorylated Akt and eNOS was evaluated by Western blot analysis. RESULTS: Brain injuries such as the cerebral infarct volume and pathological neuronal changes as well as cell apoptosis were observed in the hippocampus after hemorrhagic shock and resuscitation. Postconditioning with 2.4% sevoflurane significantly attenuated brain injuries. Wortmannin prevented the improvements of neuronal characteristics elicited by sevoflurane postconditioning as well as the hyperactivity of eNOS and phosphorylated Akt. CONCLUSIONS: Sevoflurane postconditioning could attenuate brain injury induced by hemorrhagic shock and resuscitation, and this neuroprotective effect may be partly by upregulation of eNOS through the phosphoinositide-3-kinase/Akt signaling pathway.

The Effect of Race and Shear Stress on CRP-Induced Responses in Endothelial Cells.

BACKGROUND: C-reactive protein (CRP) is an independent biomarker of systemic inflammation and a predictor of future cardiovascular disease (CVD). More than just a pure bystander, CRP directly interacts with endothelial cells to decrease endothelial nitric oxide synthase (eNOS) expression and bioactivity, decrease nitric oxide (NO) production, and increase the release of vasoconstrictors and adhesion molecules. Race is significantly associated with CRP levels and CVD risks. With aerobic exercise, the vessel wall is exposed to chronic high laminar shear stress (HiLSS) that shifts the endothelium phenotype towards an anti-inflammatory, antioxidant, antiapoptotic, and antiproliferative environment. Thus, the purpose of this study was to assess the racial differences concerning the CRP-induced effects in endothelial cells and the potential role of HiLSS in mitigating these differences. METHODS: Human umbilical vein endothelial cells (HUVECs) from four African American (AA) and four Caucasian (CA) donors were cultured and incubated under the following conditions: (1) static control, (2) CRP (10 µg/mL, 24 hours), (3) CRP receptor (FcγRIIB) inhibitor followed by CRP stimulation, (4) HiLSS (20 dyne/cm2, 24 hours), and (5) HiLSS followed by CRP stimulation. RESULTS: AA HUVECs had significantly higher FcγRIIB receptor expression under both basal and CRP incubation conditions. Blocking FcγRIIB receptor significantly attenuated the CRP-induced decrements in eNOS expression only in AA HUVECs. Finally, HiLSS significantly counteracted CRP-induced effects. CONCLUSION: Understanding potential racial differences in endothelial function is important to improve CVD prevention. Our results shed light on FcγRIIB receptor as a potential contributor to racial differences in endothelial function in AA.

Metastatic Melanoma Progression Is Associated with Endothelial Nitric Oxide Synthase Uncoupling Induced by Loss of eNOS:BH4 Stoichiometry.

Melanoma is the most aggressive type of skin cancer due to its high capability of developing metastasis and acquiring chemoresistance. Altered redox homeostasis induced by increased reactive oxygen species is associated with melanomagenesis through modulation of redox signaling pathways. Dysfunctional endothelial nitric oxide synthase (eNOS) produces superoxide anion (O2-•) and contributes to the establishment of a pro-oxidant environment in melanoma. Although decreased tetrahydrobiopterin (BH4) bioavailability is associated with eNOS uncoupling in endothelial and human melanoma cells, in the present work we show that eNOS uncoupling in metastatic melanoma cells expressing the genes from de novo biopterin synthesis pathway Gch1, Pts, and Spr, and high BH4 concentration and BH4:BH2 ratio. Western blot analysis showed increased expression of Nos3, altering the stoichiometry balance between eNOS and BH4, contributing to NOS uncoupling. Both treatment with L-sepiapterin and eNOS downregulation induced increased nitric oxide (NO) and decreased O2• levels, triggering NOS coupling and reducing cell growth and resistance to anoikis and dacarbazine chemotherapy. Moreover, restoration of eNOS activity impaired tumor growth in vivo. Finally, NOS3 expression was found to be increased in human metastatic melanoma samples compared with the primary site. eNOS dysfunction may be an important mechanism supporting metastatic melanoma growth and hence a potential target for therapy.

G protein-coupled estrogen receptor regulates the KLF2-dependent eNOS expression by activating of Ca2+ and EGFR signaling pathway in human endothelial cells.

G protein-coupled estrogen receptor (GPER) is important for maintaining normal blood vessel function by preventing endothelial cell dysfunction. It has been reported that G-1, an agonist of GPER, increases nitric oxide (NO) production through the phosphorylation of endothelial nitric oxide synthase (eNOS). However, the effect of GPER activation on eNOS expression has not been studied. Our results show that G-1 significantly increased the expression of eNOS and Kruppel-like factor 2 (KLF2) in human endothelial EA.hy926 cells. The individual silences of KLF2 and GPER attenuated G-1-induced eNOS expression. In addition, inhibition of the Gαq and Gßγ suppressed G-1-induced the expression of eNOS and KLF2 in EA.hy926 cells. Interestingly, these effects were similar in HUVECs. Furthermore, we found that GPER-mediated Ca2+ signaling increased the phosphorylation of CaMKKß, AMPK, and CaMKIIα in the cells. The phosphorylation of histone deacetylase 5 (HDAC5) by activation of AMPK and CaMKIIα increased the expression of eNOS via transcriptional activity of KLF2. We further demonstrate that GPER activation increased the phosphorylation of Src, EGFR, ERK5, and MEF2C and consequently induced the expression of eNOS and KLF2. Meanwhile, inhibition of ERK5 and HDAC5 suppressed the expression of eNOS and KLF2 induced by G-1 in the cells. These findings suggest that GPER provides a novel mechanism for understanding the regulation of eNOS expression and is an essential therapeutic target in preventing cardiovascular-related endothelial dysfunction.

Gene expression profiles during tissue remodeling following bladder outlet obstruction.

Bladder outlet obstruction (BOO) often results in lower urinary tract symptoms (LUTSs) and negatively affects quality of life. Here, we evaluated gene expression patterns in the urinary bladder during tissue remodeling due to BOO. We divided BOO model rats into two groups according to the degree of hypertrophy of smooth muscle in the bladder. The strong muscular hypertrophy group, which exhibited markedly increased bladder smooth muscle proportion and HIF1α mRNA levels compared with the control group, was considered a model for the termination of hypertrophy, whereas the mild muscular hypertrophy group was considered a model of the initiation of hypertrophy. Some genes related to urinary function showed different expression patterns between the two groups. Furthermore, we found that several genes, including D-box binding PAR bZIP transcription factor (DBP), were upregulated only in the mild muscular hypertrophy group. DBP expression levels were increased in bladder smooth muscle cells in response to hypoxic stress. DBP associated with enhancer and promoter regions of NOS3 gene locus and upregulated NOS3 gene expression under hypoxic conditions. These findings suggested that the regulatory systems of gene expression were altered during tissue remodeling following BOO. Furthermore, circadian clock components might be involved in control of urinary function via transcriptional gene regulation in response to hypoxic stimuli.

Activation of the Central Renin-Angiotensin System Causes Local Cerebrovascular Dysfunction.

Background and Purpose: Hypertension is a leading risk factor for cerebrovascular disease and loss of brain health. While the brain renin-angiotensin system (RAS) contributes to hypertension, its potential impact on the local vasculature is unclear. We tested the hypothesis that activation of the brain RAS would alter the local vasculature using a modified deoxycorticosterone acetate (DOCA) model. Methods: C57BL/6 mice treated with DOCA (50 mg SQ; or shams) were given tap H2O and H2O with 0.9% NaCl for 1 to 3 weeks. Results: In isolated cerebral arteries and parenchymal arterioles from DOCA-treated male mice, endothelium- and nitric oxide-dependent dilation was progressively impaired, while mesenteric arteries were unaffected. In contrast, cerebral endothelial function was not significantly affected in female mice treated with DOCA. In males, mRNA expression of renal Ren1 was markedly reduced while RAS components (eg, Agt and Ace) were increased in both brain and cerebral arteries with central RAS activation. In NZ44 reporter mice expressing GFP (green fluorescent protein) driven by the angiotensin II type 1A receptor (Agtr1a) promoter, DOCA increased GFP expression ≈3-fold in cerebral arteries. Impaired endothelial responses were restored to normal by losartan, an AT1R (angiotensin II type 1 receptor) antagonist. Last, DOCA treatment produced inward remodeling of parenchymal arterioles. Conclusions: These findings suggest activation of the central and cerebrovascular RAS impairs endothelial (nitric oxide dependent) signaling in brain through expression and activation of AT1R and sex-dependent effects. The central RAS may be a key contributor to vascular dysfunction in brain in a preclinical (low renin) model of hypertension. Because the brain RAS is also activated during aging and other diseases, a common mechanism may promote loss of endothelial and brain health despite diverse cause.

Chrysin attenuates high-fat-diet-induced myocardial oxidative stress via upregulating eNOS and Nrf2 target genes in rats.

Hypercholesterolemia is one of the risk factors associated with increased morbidity and mortality in cardiovascular disorders. Chrysin (Chy) is reported to exhibit anti-inflammatory, anti-cancerous, anti-oxidative, anti-aging, and anti-atherogenic properties. In the present study, we aimed to investigate whether Chy would mediate the cardioprotective effect against hypercholesterolemia-triggered myocardial oxidative stress. Male Sprague Dawley rats were divided into different groups as control and fed with high-fat diet (HFD) followed by oral administration of Chy (100 mg/kg b.wt), atorvastatin (Atv) (10 mg/kg b.wt), and L-NAME (10 mg/kg b.wt) for 30 days. At the end of the experimental period, the rats were sacrificed and tissues were harvested. Biochemical results showed a significant increase of cardiac disease marker enzymes (ALT, AST, and CKMB), lipid peroxidation, and lipid profile (TC, TG, LDL, and VLDL) in HFD-fed rat tissues when compared to control, whereas oral administration of Chy significantly reduced the activities of these marker enzymes and controlled the lipid profile. qRT-PCR studies revealed that Chy administration significantly increased the expression of endothelial nitric oxide synthase (eNOS), and Nrf2 target genes such as SOD, catalase, and GCL3 in left ventricular heart tissue of HFD-challenged rats. Immunohistochemistry results also showed that Chy treatment increased myocardial protein expression of eNOS and Nrf2 in HFD-challenged rats. Concluding the results of the present study, the Chy could mediate the cardioprotective effect through the activation of eNOS and Nrf2 signaling against hypercholesterolemia-induced oxidative stress. Thus, the administration of Chy would provide a promising therapeutic strategy for the prevention of HFD-induced oxidative stress-mediated myocardial complications.

Disrupted eNOS activity and expression account for vasodilator dysfunction in different stage of sepsis.

AIMS: Sepsis is a severe endothelial dysfunction syndrome. The role of endothelial nitric oxide synthase (eNOS) in endothelial dysfunction induced by sepsis is controversial. To explore the role of eNOS in vascular dysfunction. MAIN METHODS: The effect of sepsis on vasodilation and eNOS levels was examined in septic mouse arteries and in cell models. KEY FINDINGS: In early sepsis mouse arteries, endothelium-dependent relaxation decreased and phosphorylation of the inhibitory Thr495 site in endothelial nitric oxide synthase increased. Mechanically, the phosphorylation of endothelial nitric oxide synthase at Thr497 in bovine aortic endothelial cells occurred in a protein kinase C-α dependent manner. In late sepsis, both nitric oxide-dependent relaxation responses and endothelial nitric oxide synthase levels were decreased in septic mice arteries. Endothelial nitric oxide synthase levels expression levels decreased in tumor necrosis factor-α-treated human umbilical vein endothelial cells and this could be prevented by the ubiquitin proteasome inhibitor (MG-132). MG-132 could reverse the decrease in endothelial nitric oxide synthase expression and improve nitric oxide-dependent vasodilator dysfunction in septic mice arteries. SIGNIFICANCE: These data indicate that vasodilator dysfunction is induced by the increased phosphorylation of endothelial nitric oxide synthase in early sepsis and its degradation in late sepsis.

Maintenance of Tight Junction Integrity in the Absence of Vascular Dilation in the Brain of Mice Exposed to Ultra-High-Dose-Rate FLASH Irradiation.

Persistent vasculature abnormalities contribute to an altered CNS microenvironment that further compromises the integrity of the blood-brain barrier and exposes the brain to a host of neurotoxic conditions. Standard radiation therapy at conventional (CONV) dose rate elicits short-term damage to the blood-brain barrier by disrupting supportive cells, vasculature volume and tight junction proteins. While current clinical applications of cranial radiotherapy use dose fractionation to reduce normal tissue damage, these treatments still cause significant complications. While dose escalation enhances treatment of radiation-resistant tumors, methods to subvert normal tissue damage are clearly needed. In this regard, we have recently developed a new modality of irradiation based on the use of ultra-high-dose-rate FLASH that does not induce the classical pathogenic patterns caused by CONV irradiation. In previous work, we optimized the physical parameters required to minimize normal brain toxicity (i.e., FLASH, instantaneous intra-pulse dose rate, 6.9 · 106 Gy/s, at a mean dose rate of 2,500 Gy/s), which we then used in the current study to determine the effect of FLASH on the integrity of the vasculature and the blood-brain barrier. Both early (24 h, one week) and late (one month) timepoints postirradiation were investigated using C57Bl/6J female mice exposed to whole-brain irradiation delivered in single doses of 25 Gy and 10 Gy, respectively, using CONV (0.09 Gy/s) or FLASH (>106 Gy/s). While the majority of changes found one day postirradiation were minimal, FLASH was found to reduce levels of apoptosis in the neurogenic regions of the brain at this time. At one week and one month postirradiation, CONV was found to induce vascular dilation, a well described sign of vascular alteration, while FLASH minimized these effects. These results were positively correlated with and temporally coincident to changes in the immunostaining of the vasodilator eNOS colocalized to the vasculature, suggestive of possible dysregulation in blood flow at these latter times. Overall expression of the tight junction proteins, occludin and claudin-5, which was significantly reduced after CONV irradiation, remained unchanged in the FLASH-irradiated brains at one and four weeks postirradiation. Our data further confirm that, compared to isodoses of CONV irradiation known to elicit detrimental effects, FLASH does not damage the normal vasculature. These data now provide the first evidence that FLASH preserves microvasculature integrity in the brain, which may prove beneficial to cognition while allowing for better tumor control in the clinic.

Pinellia ternata attenuates carotid artery intimal hyperplasia and increases endothelial progenitor cell activity via the PI3K/Akt signalling pathway in wire-injured rats.

CONTEXT: Clinically, Pinellia ternata (Thunb.) Breit. (Araceae) (PT) has been widely used in the treatment of atherosclerosis and hyperlipidaemia, but the underlying mechanisms are still not clearly understood. OBJECTIVE: This research was conducted to confirm the mechanism by which PT affects carotid artery intimal hyperplasia. MATERIALS AND METHODS: An intestinal hyperplasia Sprague-Dawley rat model was established by carotid artery injury. The rats were randomly divided into five groups (n = 8): sham, model, PT (with daily intragastric administration of 10 g/mL/kg PT tubers water extract), PT+LY294002 (with intraperitoneal injection of 50 mg/kg LY294002 + 10 g/mL/kg PT) and endothelial progenitor cells (EPCs) (with injection of 5 × 105/cells), and treated for 4 or 8 weeks. RESULTS: HE staining showed that PT attenuated intimal hyperplasia. RT-PCR, Western blotting and immunohistochemistry showed that PT increased the expression of vascular endothelial growth factor (VEGF) and eNOS in the atherosclerotic carotid artery. PT increased the Dil-acLDL+/FITC-UEA-1+ population (from 0.41 ± 0.085% to 0.60 ± 0.092%) in the blood, decreased TCHO, TG, LDL-C, IL-6 and TNF-α levels, and increased HDL-C and IL-10 levels in the blood. However, these changes were reversed by the PI3K/Akt pathway inhibitor LY294002. DISCUSSION AND CONCLUSIONS: PT can be developed as an atherosclerosis and carotid intimal hyperplasia treatment drug. Therefore, further study will focus on the effects of PT on intimal hyperplasia in wire-injured atherosclerosis patients and explore in depth some other relevant molecular mechanisms.

MiR-21-5p directly contributes to regulating eNOS expression in human artery endothelial cells under normoxia and hypoxia.

Clinical conditions associated with hypoxia and oxidative stress, such as fetal growth restriction (FGR), results in endothelial dysfunction. Previous reports show that changes in eNOS expression under these conditions are tightly controlled by DNA methylation and histone posttranslational modifications. However, the contribution of an orchestrating epigenetic mechanism, such as miRNAs, on the NO-related genes expression has not been addressed. We aimed to determine the levels of miRNAs highly expressed in normal endothelial cells (EC), miR-21 and miR-126, in FGR human umbilical artery EC (HUAEC), and their effects on hypoxia-dependent regulation of both, NO-related and oxidative stress-related genes. Results were validated by transcriptome analysis of HUAEC cultured under chronic low oxygen conditions. Cultured FGR-HUAEC showed decreased hsa-miR-21, DDAH1, SOD1, and NRF2, but increased miR-126, NOX4, and eNOS levels, compared with controls. MiR-21-5p levels in FGR were associated with increased hg-miR-21 gene promoter methylation, with no changes in hg-miR-126 gene promoter methylation. HUAEC exposed to hypoxia showed a transient increase in eNOS and DDAH11, paralleled by decrease miR-21-5p levels, but no changes in miR-126-3p and the other genes under study. Transcriptome profiling showed an inverse relationship among miR-21 and several transcripts targeted by miR-21 in HUAEC exposed to hypoxia, meanwhile miR-21-5p-mimic decreased eNOS and DDAH1 transcripts stability, blocking their induction by hypoxia. Consequently, FGR programs a hypoxia-related miRNA that contributes to the regulation of the NO pathway, involving a direct effect of miR-21-5p on eNOS transcript stability, not previously reported. Moreover, hypoxia downregulates miR-21-5p, contributing to increasing the expression of NO-related genes in arterial endothelial cells.

Progesterone promotes endothelial nitric oxide synthase expression through enhancing nuclear progesterone receptor-SP-1 formation.

Progesterone exerts antihypertensive actions partially by modulating endothelial nitric oxide synthase (eNOS) activity. Here, we aimed to investigate the effects and mechanisms of progesterone on eNOS expression. First, human umbilical vein endothelial cells (HUVECs) were exposed to progesterone and then the eNOS transcription factor specificity protein-1 (SP-1) and progesterone receptor (PRA/B) expression were assessed by Western blotting and qRT-PCR. The interaction between SP-1 and PRA/B was next determined through coimmunoprecipitation assay. The chromatin immunoprecipitation assay and luciferase assay were used to investigate the relationship of PRA/B, SP-1, and eNOS promoter. At last, rats were intraperitoneally injected with progesterone receptor antagonist RU-486, and then the expression of eNOS and vasodilation function in thoracic aorta and mesenteric artery were measured. The results showed that progesterone could increase eNOS expression in HUVECs. Further study showed that progesterone increased PRA-SP-1 complex formation and facilitated PRA/B and SP-1 binding to eNOS promoter. Mutating SP-1 or PR-binding motif on eNOS promoter abolished the effect of progesterone on eNOS gene transcription. We also observed that progesterone receptor antagonist RU-486 reduced eNOS expression and impaired vasodilation in rats. Those results suggest that progesterone modulates eNOS expression through promoting PRA-SP-1 complex formation, and progesterone antagonist attenuates eNOS expression, leading to the loss of vascular relaxation.NEW & NOTEWORTHY Progesterone directly upregulated endothelial nitric oxide synthase (eNOS) expression in human endothelial cells. Progesterone augmented eNOS promoter activity through a progesterone receptor A- and specificity protein-1-dependent manner. Antagonism of the progesterone receptor reduced eNOS expression and impaired vasodilation in rats.

Circadian Rhythms of Endothelial Nitric Oxide Synthase and Toll-like Receptors 2 Production in Females with Rheumatoid Arthritis Depending on NOS3 Gene Polymorphism.

BACKGROUND: Rheumatoid Arthritis (RA) is an autoimmune polygenic disease characterized by rapid disability progression and high prevalence. Progression of RA is closely associated with chronobiological changes in the production of some hormones and inflammatory mediators, influencing the disease course and therapy efficacy. The main pathogenetic mechanism of RA is angiogenesis, which is controlled by biological clock-genes. Further investigation of circadian rhythms of angiogenic mediators production in RA patients may be considered as important and relevant. The aim of this study was to establish daily variability of serum endothelial Nitric Oxide Synthase (NOS3) and toll-like receptors 2 (sTLR2) levels in female RA patients depending on the NOS3 gene polymorphism. METHODS: We examined 173 RA patients (100% female) aged 43.7 ± 7.35 years and 34 age-matched healthy women without joint diseases and autoimmune diseases (control). RA was diagnosed by ACR/EULAR 2010 criteria. Blood serum NOS3 and sTLR2 levels were determined at 08:00 and 20:00 using Cloud-Clone Corp kits (USA). NOS3 T-786С (rs2070744) polymorphism was determined by Real-Time PCR (Bio-Rad iCycler IQ5) using SNP-express kits. The SPSS22 software package was used for statistical processing of the results. RESULTS: Females with RA demonstrated oppositely directed serum NOS3 and sTLR2 daily changes: NOS3 level in the morning (08:00) was lower than in the evening (+ 45.5 ± 30.7%), and sTLR2 level in the evening (at 20:00) was lower than in the morning (-21.6 ± 13.1%). RA patients had differences in NOS3 and sTLR2 production depending on NOS3 T786C genotype. CC subjects had NOS3 level at 08:00, 20:00 and day average levels lower (16-25%), and sTLR2 level higher (24-27%) than those of TT subjects. RA patients, carriers of CC genotype, had higher chances of NOS3 and sTLR2 aberrant production compared to TT and TC genotype carriers (OR = 2.99 and 4.79, respectively). CONCLUSION: RA patients demonstrated oppositely directed circadian changes of serum NOS3 and sTLR2. CC genotype carriers had lower NOS3 and higher sTLR2 production rates than TT and TC genotype carriers.

lncRNA ZFAS1 Improves Neuronal Injury and Inhibits Inflammation, Oxidative Stress, and Apoptosis by Sponging miR-582 and Upregulating NOS3 Expression in Cerebral Ischemia/Reperfusion Injury.

The interplay between lncRNAs and miRNAs was reported to be associated with cerebral ischemia reperfusion injury (CIRI). Besides, the lncRNA ZFAS1 was significantly downregulated in patients with ischemic stroke. However, the exact biological role of ZFAS1 and its mechanism in CIRI remain to be elucidated. The current study was designed to explore the protective effects of ZFAS1 on neuronal injury, inflammation, oxidative stress, and neuronal apoptosis in CIRI. ZFAS1 and miR-582-3p levels were assessed by RT-qPCR. Cerebral infarction was assessed by tetrazolium chloride (TTC) staining. Inflammation and oxidative stress were measured by ELISA and matched test kits. The apoptotic rate and apoptotic mediators in OGD/R-suffered PC12 cells were respectively detected by flow cytometry and western blot. NO production was measured by NO kit and western blot assay. The regulatory relationship between lncRNA ZFAS1 and miR-582-3p was analyzed using the luciferase reporter assay. ZFAS1 was downregulated in CIRI. ZFAS1 overexpression alleviated neurological function deficit and neuronal damage in a MCAO rat model. Besides, ZFAS1 upregulation or miR-582-3p downregulation could both inhibit the inflammation, oxidative stress, and apoptosis and enhance NO level in OGD/R-suffered PC12 cells. Reporter assay indicated that ZFAS1 served as a molecular sponge for miR-582-3p. In addition, ZFAS1 could negatively regulate miR-582-3p expression and release its effects in CIRI. Taken together, our study revealed that lncRNA ZFAS1 protected against neuronal damage and modulated the inflammation, oxidative stress, apoptosis, and NO level via regulating miR-582-3p in cerebral I/R injury. Therefore, lncRNA ZFAS1 might serve as a therapeutic application to suppress CIRI progression.

Oxidative Stress and Mitochondrial Abnormalities Contribute to Decreased Endothelial Nitric Oxide Synthase Expression and Renal Disease Progression in Early Experimental Polycystic Kidney Disease.

Vascular abnormalities are the most important non-cystic complications in Polycystic Kidney Disease (PKD) and contribute to renal disease progression. Endothelial dysfunction and oxidative stress are evident in patients with ADPKD, preserved renal function, and controlled hypertension. The underlying biological mechanisms remain unknown. We hypothesized that in early ADPKD, the reactive oxygen species (ROS)-producing nicotinamide adenine dinucleotide phosphate hydrogen (NAD(P)H)-oxidase complex-4 (NOX4), a major source of ROS in renal tubular epithelial cells (TECs) and endothelial cells (ECs), induces EC mitochondrial abnormalities, contributing to endothelial dysfunction, vascular abnormalities, and renal disease progression. Renal oxidative stress, mitochondrial morphology (electron microscopy), and NOX4 expression were assessed in 4- and 12-week-old PCK and Sprague-Dawley (wild-type, WT) control rats (n = 8 males and 8 females each). Endothelial function was assessed by renal expression of endothelial nitric oxide synthase (eNOS). Peritubular capillaries were counted in hematoxylin-eosin (H&E)-stained slides and correlated with the cystic index. The enlarged cystic kidneys of PCK rats exhibited significant accumulation of 8-hydroxyguanosine (8-OHdG) as early as 4 weeks of age, which became more pronounced at 12 weeks. Mitochondria of TECs lining cysts and ECs exhibited loss of cristae but remained preserved in non-cystic TECs. Renal expression of NOX4 was upregulated in TECs and ECs of PCK rats at 4 weeks of age and further increased at 12 weeks. Contrarily, eNOS immunoreactivity was lower in PCK vs. WT rats at 4 weeks and further decreased at 12 weeks. The peritubular capillary index was lower in PCK vs. WT rats at 12 weeks and correlated inversely with the cystic index. Early PKD is associated with NOX4-induced oxidative stress and mitochondrial abnormalities predominantly in ECs and TECs lining cysts. Endothelial dysfunction precedes capillary loss, and the latter correlates with worsening of renal disease. These observations position NOX4 and EC mitochondria as potential therapeutic targets in PKD.

Atorvastatin alleviates left ventricular remodeling in isoproterenol-induced chronic heart failure in rats by regulating the RhoA/Rho kinase signaling pathway.

BACKGROUND: Chronic heart failure (CHF) is characterized by left ventricular dysfunction and altered autonomic control of cardiac function. This study aimed to investigate the effects of atorvastatin on left ventricular remodeling (LVR) and cardiac function in rats with isoproterenol-induced CHF and the possible mechanism. METHODS: An isoproterenol-induced CHF model was established in rata, which were subsequently treated with atorvastatin. Echocardiography, hemodynamic, and left ventricular mass indexes were assessed. The mRNA expression of RhoA, Rho kinase, and endothelial nitric oxide synthase (eNOS) was determined by RT-qPCR. The protein expression of myosin-binding subunit (MBS), MBS-P, eNOS, phosphorylated-eNOS, RhoA, and Rho kinase was measured by Western blot analysis. The relative activity of NADPH oxidase, ROS, and NO was assessed by ELISA. RESULTS: Isoproterenol-induced CHF rats treated with atorvastatin exhibited decreased left ventricular end-systolic dimension, left ventricular end-diastolic dimension, left ventricular end-diastolic pressure, left ventricular mass index, maximum fall rate of change in left ventricular pressure, heart rate (p < 0.001), expression of RhoA, Rho kinase, MBS and MBS-P (p < 0.01), and relative activity of NADPH oxidase, ROS and NO (p < 0.05) and increased left ventricular short axis fractional shortening, left ventricular end-systolic pressure, maximum rise rate of change in left ventricular pressure (p < 0.001) and expression of eNOS, and phosphorylated-eNOS ser1177 (all p < 0.05) compared with those of rats with isoproterenol-induced CHF. CONCLUSION: We demonstrated that atorvastatin inhibits LVR and improves cardiac function in rats with isoproterenol-induced CHF through inhibition of the RhoA/Rho kinase signaling pathway.

Use of embryonic stem cell-derived cardiomyocytes to study cardiotoxicity of bisphenol AF via the GPER/CAM/eNOS pathway.

Bisphenol AF (BPAF) is a derivative of bisphenol A (BPA) that is widely used in fluorinated polymers, fluorinated rubber, electronic equipment, plastic optical fibers, etc. Studies have shown that BPAF exposure is associated with a number of diseases; however, little is known about the effects of BPAF on cardiomyocytes. We investigated the impact of chronic exposure to BPAF on cardiomyocytes derived from embryonic stem cells (ESCs). The present study showed that chronic exposure to various concentrations of BPAF (0, 8, 200 and 1000 ng/ml) induces cardiomyocyte hypertrophy. The ratios of microfilaments to mitochondrial length and the ratio of microfilaments to cell nuclei and MYH7b levels indicate that BPAF exposure alters the morphology of the cells and mitochondria. Furthermore, BPAF exposure at concentrations from 8 to 1000 ng/ml results in an increase in G protein-coupled estrogen receptor (GPER) expression. Additionally, our results suggest that these effects of BPAF mediate cardiomyocyte hypertrophy apparently due to an increase in the production of reactive nitrogen species (RNS) via an increase in endothelial NO synthase (eNOS). These results imply that ESC-based myocardial differentiation can be an excellent cellular model to study BPAF-induced cardiotoxicity at the cellular and molecular levels.

Spontaneous Pulmonary Hypertension Associated With Systemic Sclerosis in P-Selectin Glycoprotein Ligand 1-Deficient Mice.

OBJECTIVE: Pulmonary arterial hypertension (PAH), one of the major complications of systemic sclerosis (SSc), is a rare disease with unknown etiopathogenesis and noncurative treatments. As mice deficient in P-selectin glycoprotein ligand 1 (PSGL-1) develop a spontaneous SSc-like syndrome, we undertook this study to analyze whether they develop PAH and to examine the molecular mechanisms involved. METHODS: Doppler echocardiography was used to estimate pulmonary pressure, immunohistochemistry was used to assess vascular remodeling, and myography of dissected pulmonary artery rings was used to analyze vascular reactivity. Angiotensin II (Ang II) levels were quantified by enzyme-linked immunosorbent assay, and Western blotting was used to measure Ang II type 1 receptor (AT1 R), AT2 R, endothelial cell nitric oxide synthase (eNOS), and phosphorylated eNOS expression in lung lysates. Flow cytometry allowed us to determine cytokine production by immune cells and NO production by endothelial cells. In all cases, there were 4-8 mice per experimental group. RESULTS: PSGL-1-/- mice showed lung vessel wall remodeling and a reduced mean ± SD expression of pulmonary AT2 R (expression ratio [relative to ß-actin] in female mice age >18 months: wild-type mice 0.799 ± 0.508 versus knockout mice 0.346 ± 0.229). With aging, female PSGL-1-/- mice had impaired up-regulation of estrogen receptor α (ERα) and developed lung vascular endothelial dysfunction coinciding with an increase in mean ± SEM pulmonary Ang II levels (wild-type 48.70 ± 5.13 pg/gm lung tissue versus knockout 78.02 ± 28.09 pg/gm lung tissue) and a decrease in eNOS phosphorylation, leading to reduced endothelial NO production. These events led to a reduction in the pulmonary artery acceleration time:ejection time ratio in 33% of aged female PSGL-1-/- mice, indicating pulmonary hypertension. Importantly, we found expanded populations of interferon-γ-producing PSGL-1-/- T cells and B cells and a reduced presence of regulatory T cells. CONCLUSION: The absence of PSGL-1 induces a reduction in Treg cells, NO production, and ERα expression and causes an increase in Ang II in the lungs of female mice, favoring the development of PAH.

Titanate nanotubes at non-cytotoxic concentrations affect NO signaling pathway in human umbilical vein endothelial cells.

Titanate nanotubes (TiNTs) have been considered as biocompatible nanomaterials (NMs) for biomedical uses. Hereby, we compared the toxicity of TiNTs and TiO2 nanoparticles (NPs) to human umbilical vein endothelial cells (HUVECs). Our results showed that TiNTs were less effectively internalized into HUVECs compared with TiO2 NPs, but none of the NMs induced cytotoxicity or activation of endoplasmic reticulum (ER) stress biomarkers. In addition, intracellular reactive oxygen species (ROS) was only modestly induced by TiNTs and TiO2 NPs. However, both types of NMs significantly promoted the protein levels of vascular cell adhesion molecule-1 (VCAM-1). TiNTs also promoted the release of soluble (sVCAM-1), but THP-1 monocyte adhesion onto HUVECs was only induced by TiO2 NPs. TiNTs decreased the production of NO, associated with a decrease of protein levels of endothelial NO synthase (eNOS). The transcription factors of eNOS, including kruppel-like factor 2 (KLF2) and KLF4, were more effectively down-regulated by TiNTs compared with TiO2 NPs. In conclusion, our results indicated that TiNTs, albeit not cytotoxic, might impair NO signaling pathway in human endothelial cells leading to the activation of endothelial cells.

Mid-dose losartan mitigates diabetes-induced hepatic damage by regulating iNOS, eNOS, VEGF, and NF-κB expressions

Background/aim: Losartan, an antihypertensive drug, is highly preferred in patients with diabetes mellitus (DM) and hypertension because of its retarding effect on diabetic nephropathy. In this study, we investigated the potential therapeutic effect of different doses of losartan on hepatic damage in a streptozotocin (STZ, 50 mg/kg)-induced DM model in rats. Materials and methods: In this study, five different groups were formed: control, DM, low-dose losartan (5 mg/kg), mid-dose losartan (20 mg/kg), and high-dose losartan (80 mg/kg). Liver tissues of experimental groups were evaluated immunohistochemically for TUNEL, iNOS, eNOS, VEGF, and NF-κB pathways. In addition to immunohistochemical analysis, analyses of SOD and MDA, which are oxidative stress markers, were also performed and the results were evaluated together. Results: When biochemical and immunohistochemical findings were evaluated together, it was found that the results obtained from the mid-dose losartan group were closer to those of the control than the other groups. Conclusion: This study indicated that mid-dose losartan administration may have a therapeutic effect by inhibiting apoptosis and regulating iNOS, eNOS, VEGF, and NF-κB protein expressions in DM-induced hepatic damage.