eNOS KO Research Articles

Structural brain abnormalities in endothelial nitric oxide synthase-deficient mice revealed by high-resolution magnetic resonance imaging.

Endothelial nitric oxide synthase (eNOS) produces nitric oxide, which is essential for a variety of physiological functions in the brain. Previous work has demonstrated the detrimental effects of eNOS deficiency on brain function in male eNOS knockout (eNOS KO) mice. However, the effect of eNOS deficiency on brain structure and any association between these effects and sex is unknown. This study used three-dimensional high-resolution ex vivo magnetic resonance imaging and behavioral tests of anxiety and cognitive performance to investigate structure-function relationships in the brain of female and male eNOS KO mice in young adulthood. While there were no differences in anxiety-like behavior or locomotion, there was a sex-specific deficit in contextual fear memory retention in male, but not in female, eNOS mice compared to wild-type controls. Moreover, we found that eNOS deficiency induced changes in multiple brain regions that are involved in learning and fear memory including the hippocampus, amygdala, hypothalamus, and areas of the cortex. Several of these MRI-detectable neuroanatomical changes were dependent on sex. The observation that eNOS deficiency impacts brain structure at an early age demonstrates the importance of eNOS for healthy brain development.

Integrin-Linked Kinase Expression in Human Valve Endothelial Cells Plays a Protective Role in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is highly prevalent during aging. CAVD initiates with endothelial dysfunction, leading to lipid accumulation, inflammation, and osteogenic transformation. Integrin-linked kinase (ILK) participates in the progression of cardiovascular diseases, such as endothelial dysfunction and atherosclerosis. However, ILK role in CAVD is unknown. First, we determined that ILK expression is downregulated in aortic valves from patients with CAVD compared to non-CAVD, especially at the valve endothelium, and negatively correlated with calcification markers. Silencing ILK expression in human valve endothelial cells (siILK-hVECs) induced endothelial-to-mesenchymal transition (EndMT) and promoted a switch to an osteoblastic phenotype; SiILK-hVECs expressed increased RUNX2 and developed calcified nodules. siILK-hVECs exhibited decreased NO production and increased nitrosative stress, suggesting valvular endothelial dysfunction. NO treatment of siILK-hVECs prevented VEC transdifferentiation, while treatment with an eNOS inhibitor mimicked ILK-silencing induction of EndMT. Accordingly, NO treatment inhibited VEC calcification. Mechanistically, siILK-hVECs showed increased Smad2 phosphorylation, suggesting a TGF-β-dependent mechanism, and NO treatment decreased Smad2 activation and RUNX2. Experiments performed in eNOS KO mice confirmed the involvement of the ILK-eNOS signaling pathway in valve calcification, since aortic valves from these animals showed decreased ILK expression, increased RUNX2, and calcification. Our study demonstrated that ILK endothelial expression participates in human CAVD development by preventing endothelial osteogenic transformation.

Endothelial nitric oxide deficiency results in abnormal placental metabolism

Placental metabolism determines the amount of nutrients available to the fetus and may be altered in pregnancies complicated by fetal growth restriction (FGR). To study which metabolites are associated with FGR, we performed 1H high-resolution magic angle spinning magnetic resonance spectroscopy of placental tissue from endothelial nitric oxide synthase knockout (eNOS KO) mice, a model of FGR, and C57BL/6J controls at embryonic day 17.5 (n = 24/genotype). The relative concentration of glucose was increased in the placentas of eNOS KO mice compared to controls (p = 0.006). This study highlights the potential for glucose as a biomarker of abnormal placental metabolism that leads to FGR.

Pleiotropic activation of endothelial function by angiotensin II receptor blockers is crucial to their protective anti-vascular remodeling effects

There are no therapeutics that directly enhance chronic endothelial nitric oxide (NO) release, which is typically associated with vascular homeostasis. In contrast, angiotensin II (AngII) receptor type 1 (AT1R) blockers (ARBs) can attenuate AngII-mediated oxidative stress, which often leads to increased endothelial NO bioavailability. Herein, we investigate the potential presence of direct, AngII/AT1R-independent ARB class effects on endothelial NO release and how this may result in enhanced aortic wall homeostasis and endothelial NO-specific transcriptome changes. Treatment of mice with four different ARBs induced sustained, long-term inhibition of vascular contractility by up to 82% at 16 weeks and 63% at 2 weeks, an effect reversed by L-NAME and absent in endothelial NO synthase (eNOS) KO mice or angiotensin converting enzyme inhibitor captopril-treated animals. In absence of AngII or in tissues with blunted AT1R expression or incubated with an AT2R blocker, telmisartan reduced vascular tone, supporting AngII/AT1R-independent pleiotropism. Finally, telmisartan was able to inhibit aging- and Marfan syndrome (MFS)-associated aortic root widening in NO-sensitive, BP-independent fashions, and correct aberrant TGF-β signaling. RNAseq analyses of aortic tissues identified early eNOS-specific transcriptome reprogramming of the aortic wall in response to telmisartan. This study suggests that ARBs are capable of major class effects on vasodilatory NO release in fashions that may not involve blockade of the AngII/AT1R pathway. Broader prophylactic use of ARBs along with identification of non-AngII/AT1R pathways activated by telmisartan should be investigated.

Sildenafil attenuates intestinal injury in necrotizing enterocolitis independently of endothelial nitric oxide synthase

BackgroundNecrotizing enterocolitis (NEC) is a devastating disease that impacts the intestine of premature infants. Sildenafil has shown benefit in colitis and ischemia/reperfusion models but has not been adequately studied in NEC. Sildenafil's best studied mechanism involves augmenting nitric oxide induced vasodilation. We hypothesized that sildenafil would improve outcomes during experimental NEC in an eNOS dependent manner. MaterialsNEC was induced in five-day old mouse pups with gavage formula feeds plus intermittent hypoxia and hypothermia. Using wild type (WT) mice, the route of sildenafil administration was studied in the following groups: (1) breastfed controls, (2) NEC + oral (PO) sildenafil, (3) NEC + PO vehicle, (4) NEC + intraperitoneal (IP) sildenafil, (5) NEC + IP vehicle. The eNOS KO groups studied included: (1) breastfed controls, (2) NEC + PO sildenafil, (3) NEC + PO vehicle. Data were tested for normality and compared using t-tests or Mann-Whitney with a p-value <0.05 considered significant. ResultsIn WT mice, oral and IP sildenafil resulted in improved clinical outcomes compared to their respective vehicle group. Only orally administered sildenafil significantly improved perfusion to the intestine and protected it from macroscopic and histologic injury. When repeated in eNOS KO mice, oral sildenafil improved clinical scores and attenuated intestinal injury scores, despite no effect on intestinal perfusion. ConclusionsSildenafil, when administered orally, improves clinical outcomes and protects the intestine in a murine model of experimental necrotizing enterocolitis. While sildenafil requires eNOS to impact mesenteric perfusion, it does not appear to be dependent on eNOS to attenuate intestinal injury.

Endothelial Dysfunction Accelerates Impairment of Mitochondrial Function in Ageing Kidneys via Inflammasome Activation.

Chronic kidney disease is a common problem in the elderly and is associated with increased mortality. We have reported on the role of nitric oxide, which is generated from endothelial nitric oxide synthase (eNOS), in the progression of aged kidneys. To elucidate the role of endothelial dysfunction and the lack of an eNOS-NO pathway in ageing kidneys, we conducted experiments using eNOS and ASC-deficient mice. C57B/6 J mice (wild type (WT)), eNOS knockout (eNOS KO), and ASC knockout (ASC KO) mice were used in the present study. Then, eNOS/ASC double-knockout (eNOS/ASC DKO) mice were generated by crossing eNOS KO and ASC KO mice. These mice were sacrificed at 17−19 months old. The Masson positive area and the KIM-1 positive area tended to increase in eNOS KO mice, compared with WT mice, but not eNOS/ASC DKO mice. The COX-positive area was significantly reduced in eNOS KO mice, compared with WT and eNOS/ASC DKO mice. To determine whether inflammasomes were activated in infiltrating macrophages, the double staining of IL-18 and F4/80 was performed. IL-18 and F4/80 were found to be co-localised in the tubulointerstitial areas. Inflammasomes play a pivotal role in inflammaging in ageing kidneys. Furthermore, inflammasome activation may accelerate cellular senescence via mitochondrial dysfunction. The importance of endothelial function as a regulatory mechanism suggests that protection of endothelial function may be a potential therapeutic target.

Sphingosine 1-phosphate-induced nitric oxide production simultaneously controls endothelial barrier function and vascular tone in resistance arteries

The regulations of endothelial permeability and vascular tone by sphingosine 1-phosphate (S1P) have been well-studied independently. Little is known about whether the effects of S1P on endothelial permeability can directly influence vascular tone in resistance arteries, which impact blood flow. The endothelium forms a partial barrier that regulates access of circulating agonists to underlying vascular smooth muscle cells (VSMCs). We hypothesized that physiological concentrations of circulating S1P simultaneously control endothelial barrier function and vascular tone through endothelial production of nitric oxide (NO). We adapted the pressure myograph system to simultaneously measure both functions in pressurized mesenteric compared to uterine resistance arteries from wild-type and eNOS KO mice. We established that: 1) S1P interacting directly with the endothelium inside pressurized arteries generates NO that limits endothelial permeability; 2) an intact endothelium forms a partial physical barrier that regulates access of intraluminal S1P to the underlying VSMCs and 3) S1P infused lumenally also generates NO through eNOS that counterbalances the constriction induced by S1P that is able to access VSMCs and this is critical to control vascular tone. We conclude that targeting the S1P signaling system, particularly the capacity to produce NO could be clinically important in the treatment of vascular diseases.

Activation of soluble guanylyl cyclase signalling with cinaciguat improves impaired kidney function in diabetic mice.

Diabetic nephropathy is the leading cause for end-stage renal disease worldwide. Until now, there is no specific therapy available. Standard treatment with inhibitors of the renin-angiotensin system just slows down progression. However, targeting the NO/sGC/cGMP pathway using sGC activators does prevent kidney damage. Thus, we investigated if the sGC activator cinaciguat was beneficial in a mouse model of diabetic nephropathy, and we analysed how mesangial cells (MCs) were affected by related conditions in cell culture. Type 1 diabetes was induced with streptozotocin in wild-type and endothelial NOS knockout (eNOS KO) mice for 8 or 12 weeks.. Half of these mice received cinaciguat in their chow for the last 4 weeks. Kidneys from the diabetic mice were analysed with histochemical assays and by RT-PCR and western blotting.. Additionally, primary murine MCs under diabetic conditions were stimulated with 8-Br-cGMP or cinaciguat to activate the sGC/cGMP pathway. The diabetic eNOS KO mice developed most characteristics of diabetic nephropathy, most marked at 12 weeks. Treatment with cinaciguat markedly improved GFR, serum creatinine, mesangial expansion and kidney fibrosis in these animals. We determined expression levels of related signalling proteins. Thrombospondin 1, a key mediator in kidney diseases, was strongly up-regulated under diabetic conditions and this increase was suppressed by activation of sGC/cGMP signalling. Activation of the NO/sGC/PKG pathway with cinaciguat was beneficial in a model of diabetic nephropathy. Activators of sGC might be an appropriate therapy option in patients with Type 1 diabetes. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Reactivation of eNOS in the endothelium rescues eNOS KO mice from endothelial dysfunction and hypertension

Production of nitric oxide (NO) by endothelial NO synthase (eNOS; NOS3) plays a key role in regulation of vascular tone and blood pressure. The aim of this study was to establish “loss” and “gain” of function models for eNOS by using tissue specific Cre‐induced gene inactivation or reactivation. To accomplish this, two independent genotypes were created: 1) a conventional floxed eNOS (eNOSflox/flox) against exon 2 or 2) a Cre‐inducible eNOS knock‐in (KI) gene construct (eNOSinv/inv) allowing targeted removal or reactivation of eNOS expression. Both the eNOSflox/flox and eNOSinv/inv were crossed with endothelial cell (EC)‐specific tamoxifen (TAM)‐inducible Cre mice to create EC‐specific knock‐out (EC eNOS KO) and EC eNOS KI mice. We found that Cre‐specific DNA recombination occurred specifically in endothelium of both EC eNOS KO and EC eNOS KI, as assessed by quantitative polymerase chain reaction (qPCR). TAM treatment fully abolished mRNA and protein expression of eNOS in the aorta of EC eNOS KO (eNOSflox/flox Crepos) mice, but not in Cre negative or untreated mice, as determined by retro transcription qPCR and western blot analysis. Functionally, the EC eNOS KO demonstrated inhibition of Ach‐induced vasodilation of aortic rings ex vivo, as well as flow‐mediated dilation (FMD) of the arteria iliaca in vivo (assessed by ultrasound). Moreover, absence of eNOS in the endothelium of EC eNOS KO was correlated with spontaneous hypertension, assessed by Millar catheter (MAP was 80.37 ± 2.135 mmHg in EC eNOS KO against 67.88 ± 2.723 mmHg in control mice). Next, we tested blood pressure of eNOSinv/inv which fully lacked eNOS expression in all tissues and was hypertensive. In EC eNOS KI (eNOSinv/inv Crepos treated with TAM) the expression of eNOS was completely restored in endothelium, leading to a significant decrease of the blood pressure as compared to eNOSinv/inv Creneg mice. In conclusion, we report the first inducible “loss” and “gain” of function models of eNOS in the vascular endothelium demonstrating that reactivation of eNOS rescues from endothelial dysfunction and hypertension. These mice models can further be applied to non endothelial tissues to further investigate the role of eNOS in different tissues in modulating blood pressure and systemic hemodynamics.Support or Funding InformationThis work was supported by the German Research Council (DFG CO 1305/2‐1 to M.M.C.‐K.; F.L. is a scholar of the IRTG1902 to M.M.C.‐K. and B.E.I. and by a research grant of the Forschungskommission to M.M.C.‐K., Medical Faculty, Heinrich Heine University of Düsseldorf.

Acute Hypoglycemia Induces Mitochondrial Dysfunction in the Isolated Brain Microvessels: Possible Role of Endothelial Nitric Oxide Synthase

BackgroundInsulin therapy for patients with diabetes mellitus (DM) carries the risk for acute hypoglycemia (HG). Recurrent HG increases the ischemic brain injury in experimental stroke. Furthermore, HG has been shown to affect mitochondrial function in the cultured endothelial cells. Previously, we have shown that vascular mitochondria regulate endothelial nitric oxide synthase activity (eNOS). Therefore, we hypothesized that exposure to acute insulin‐induced HG in vivo negatively affects the ex vivo cerebral microvascular respiratory function. Here, we investigated for the first time, the effect of acute HG on mitochondrial respiratory function ex vivo in the isolated brain microvessels from wild‐type and endothelial nitric oxide synthase (eNOS) knock‐out mice.MethodsThree month‐old C57bl6 mice (n=6) were injected intraperitoneally (i.p) with insulin (3 U/ Kg body weight) to induce and maintain hypoglycemic state (blood glucose level &lt; 70mg/dL) for 90 minutes. In order to evaluate the HG‐independent effects of hyperinsulinemia, a group of mice (n=6) were maintained at normoglycemia after the insulin injection by i.p injection of glucose. Control mice (n=6) were injected with saline. Brain parenchymal microvessles (40μ to 300μ diameter) were isolated and oxygen consumption rate (OCR) and mitochondrial respiratory parameters were measured using Agilent Seahorse XFe24 analyzer. Basal and maximal respiration were measured along with the spare respiratory capacity, proton leak and the non‐mitochondrial respiration. Parameters were reported as OCR normalized to the protein concentration of cell lysates in each well in picomoles/L of O2/μg protein. Similar studies were also conducted in eNOS KO mice.ResultsThe maximal respiration was decreased by 47.3% (3.0±0.4 vs 5.7±0.7, p=0.01, n=6) in the isolated mouse brain microvessels from mice exposed to HG compared to saline exposed animals. Furthermore, spare respiratory capacity decreased by 76% (0.3±0.1 vs 1.3±0.1, p=0.0004, n=6) along with the non‐mitochondrial respiration (1.0±0.2 vs 2.5±0.3, p=0.01, n=6). Basal respiration and proton leak showed a strong trend towards decrease by 32.5% (2.9±0.5 vs 4.3±0.5, p=0.06, n=6) and 28% (1.8±0.3 vs 2.5±0.3, p=0.12, n=6) respectively along with the ATP production (1.4±0.2 vs 1.8±0.2, p=0.24, n=6) in microvessels from mice exposed to HG. Interestingly, similar mitochondrial respiratory changes were not observed in brain microvessels from the mice that were maintained under normal glucose levels after the insulin injection, indicating that the observed changes were solely due to the HG. Interestingly, brain microvessels from eNOS knock‐out mice exposed to HG failed to exhibit the impairments of mitochondrial respiratory function observed in wild type mice.ConclusionAcute HG induces mitochondrial respiratory dysfunction in the cerebral microvasculature. eNOS might play a key role in mediating the observed effects.Support or Funding InformationSupport: National Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A14340 Endothelial dysfunction exacerbates renal tubular cell injury through inflammasome activation in hypertensive model mice

Objectives: Hypertension causes vascular endothelial dysfunction. Inflammasome activation plays an important role in the development of kidney disease. However the relationship between endothelial function and inflammasome activation is unknown. In this study, we focused on nitric oxide (NO) as a regulator of inflammasome activation. The hypothesis was that endothelium-derived NO would inhibit inflammasome activation and control chronic inflammation in the hypertensive kidney diseases. Methods: Aldosterone (Ald) was continuously administered to wild-type mice (WT) and endothelial NO synthase-knockout (eNOS KO) mice for 4 weeks. To detect inflammasome activation, bone marrow-derived macrophages (BMDMs) were primed with LPS. After LPS priming, the BMDMs were stimulated with ATP to activate the NLRP3 inflammasome. BMDMs were simultaneously primed with GSNO. To determine the role of inflammasome activation in endothelial dysfunction, eNOS/ASC double KO mice (eNOS/ASC DKO) were generated and treated with Ald. Results: The expression of NLRP3 inflammasome-related genes was much higher in the eNOS KO-Ald group than WT-Ald group. Tubular injury and fibrosis were accelerated in the eNOS KO-Ald group as compared with the WT-Ald group. ATP stimulation and LPS priming caused NLRP3 inflammasome-dependent cell death and interleukin-1β (IL-1β) secretion, and GSNO inhibited IL-1β secretion. These data suggested that NO suppressed NLRP3 inflammasome activation directly in macrophages. Ald treatment resulted in a significant increase in the expression of inflammasome-related genes, in addition to increased tubular injury and fibrosis. In contrast, these changes were significantly suppressed in the eNOS/ASC DKO group. Conclusion: NO derived from eNOS inhibited inflammasome activation in an Ald-induced hypertensive model.

Abstract TP428: The Beneficial Effects of Chronic Remote Ischemic Conditioning in a VCID Mouse Model are eNOS Dependent

Background and Purpose: Chronic remote ischemic conditioning (C-RIC) is effective at improving cerebral blood flow (CBF) inducing vascular remodeling, and improving cognition in a bilateral carotid artery stenosis (BCAS) mouse model, a model for Vascular Cognitive Impairment and Dementia (VCID). This improvement is associated with elevations of plasma nitrite. Our aim was to determine if the beneficial effect of C-RIC was eNOS dependent. Methods: Microcoil (01.8 mm) induced BCAS model was used to induce chronic hypoperfusion. Adult eNOS-KO male mice (5-6 months) were randomly assigned to 2-groups (N=5): (1) BCAS and (2) BCAS+RIC. RIC was started 7d post-surgery daily for 3 weeks. Behavioral test and CBF was performed before termination. Functional outcomes were assessed using novel object recognition (NOR) test for non-spatial working memory, and hanging wire and beam walk test for motor/muscular impairment. Mice were followed for 4 weeks. Results: C-RIC-therapy for 3 weeks did not improve CBF in the BCAS+RIC groups at either a 2 nd weeks or 4 th weeks compared to BCAS-Sham RIC groups. There was no significant change between the BCAS and BCAS+RIC groups in the discrimination index as determined by the NOR test or poor motor function as determined by hanging wire and beam walk test. Conclusions: The beneficial effect of C-RIC in the BCAS model is abrogated in eNOS KO mice indicating that the effect of C-RIC is eNOS dependent.

Nrf2 Deficiency Unmasks the Significance of Nitric Oxide Synthase Activity for Cardioprotection.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch that controls the expression of antioxidant and cytoprotective enzymes, including enzymes catalyzing glutathione de novo synthesis. In this study, we aimed to analyze whether Nrf2 deficiency influences antioxidative capacity, redox state, NO metabolites, and outcome of myocardial ischemia reperfusion (I/R) injury. In Nrf2 knockout (Nrf2 KO) mice, we found elevated eNOS expression and preserved NO metabolite concentrations in the aorta and heart as compared to wild types (WT). Unexpectedly, Nrf2 KO mice have a smaller infarct size following myocardial ischemia/reperfusion injury than WT mice and show fully preserved left ventricular systolic function. Inhibition of NO synthesis at onset of ischemia and during early reperfusion increased myocardial damage and systolic dysfunction in Nrf2 KO mice, but not in WT mice. Consistent with this, infarct size and diastolic function were unaffected in eNOS knockout (eNOS KO) mice after ischemia/reperfusion. Taken together, these data suggest that eNOS upregulation under conditions of decreased antioxidant capacity might play an important role in cardioprotection against I/R. Due to the redundancy in cytoprotective mechanisms, this fundamental antioxidant property of eNOS is not evident upon acute NOS inhibition in WT mice or in eNOS KO mice until Nrf2-related signaling is abrogated.

Umbilical mesenchymal stromal cells provide intestinal protection through nitric oxide dependent pathways

BackgroundUmbilical-derived mesenchymal stromal cells (USCs) have shown promise in the protection of ischemic organs. We hypothesized that USCs would improve mesenteric perfusion, preserve intestinal histological architecture, and limit inflammation by nitric oxide–dependent mechanisms following intestinal ischemia/reperfusion (IR) injury. MethodsAdult wild-type C57BL/6J (WT) and endothelial nitric oxide synthase knock out (eNOS KO) mice were used: (1) WT IR + vehicle, (2) WT IR + USC, (3) eNOS KO IR + vehicle, and (4) eNOS KO IR + USC. Mice were anesthetized, and a midline laparotomy was performed. The superior mesenteric artery was clamped with a nonoccluding clamp for 60-min. Following IR, mice were treated with an injection of 250 μL phosphate buffered saline or 2 × 106 USCs suspended in 250-μL phosphate buffered saline solution. Mesenteric perfusion images were acquired using laser Doppler imaging. Perfusion was analyzed as a percentage of baseline. At 24 h, mice were euthanized, and intestines were harvested. Intestines were evaluated for injury, and data were analyzed using the Mann–Whitney or Kruskal–Wallis tests. ResultsIntestinal mesenteric perfusion was significantly improved in WT mice treated with USC therapy compared with eNOS KOs. Intestinal histological architecture was preserved with USC therapy in WT mice. However, in eNOS KO mice, this benefit was abolished. Finally, the presence of several cytokines and growth factors were significantly improved in WT mice compared with eNOS KO mice treated with USCs. ConclusionsThe benefits of USC-mediated therapy following intestinal IR injury likely occur via nitric oxide–dependent pathways. Further studies are required to define the molecular mechanisms by which USCs activate endothelial nitric oxide synthase to bring about their protective effects.

Pyruvate kinase M2 activation may protect against the progression of diabetic glomerular pathology and mitochondrial dysfunction.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (ł50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1α mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

Tyrosine phosphorylation of eNOS regulates myocardial survival after an ischaemic insult: role of PYK2.

Endothelial nitric oxide (NO) synthase (eNOS) is known to play a cardioprotective protective. However, the molecular mechanisms regulating eNOS activity during ischaemia/reperfusion (I/R) injury are incompletely understood. eNOS is a substrate for several kinases that positively or negatively affect its enzymatic activity. Herein, we sought to correlate eNOS phosphorylation status with cardiomyocyte survival and we investigated the contribution of the proline-rich tyrosine kinase 2 (PYK2)/eNOS axis to the regulation of myocardial infarct size in vivo. Exposure of H9c2 cardiomyocytes to H2O2 lead to PYK2 phosphorylation on its activator site (Y402) and eNOS phosphorylation on the inhibitor site Y656 and the activator site S1176. Both H2O2-induced eNOS phosphorylation events were abolished by PYK2 pharmacological inhibition or gene knockdown. Activity assays demonstrated that phosphorylation of the tyrosine inhibitory site exerts a dominant effect over S1176. In cardiomyocytes subjected to oxidative stress or oxygen-glucose deprivation, inhibition of PYK2 limited cell injury; this effect was prevented by inhibition of NO production. In vivo, ischaemia-reperfusion induced an early activation of PYK2, leading to eNOS phosphorylation on Y656, which, in turn, reduced NO output, as judged by the low tissue levels of its downstream effector cGMP. Moreover, pharmacological blockade of PYK2 alleviated eNOS inhibition and prevented cardiac damage following I/R injury in wild-type, but not in eNOS KO mice. The current studies demonstrate that PYK2 is a pivotal regulator of eNOS function in myocardial infarction and identify PYK2 as a novel therapeutic target for cardioprotection.

Hydrogen sulfide improves intestinal recovery following ischemia by endothelial nitric oxide-dependent mechanisms.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter that has vasodilatory properties. It may be a novel therapy for intestinal ischemia-reperfusion (I/R) injury. We hypothesized that 1) H2S would improve postischemic survival, mesenteric perfusion, mucosal injury, and inflammation compared with vehicle and 2) the benefits of H2S would be mediated through endothelial nitric oxide. C57BL/6J wild-type and endothelial nitric oxide synthase knockout (eNOS KO) mice were anesthetized, and a midline laparotomy was performed. Intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using laser Doppler. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery. Following ischemia, the clamp was removed, and the intestines were allowed to recover. Either sodium hydrosulfide (2 nmol/kg or 2 µmol/kg NaHS) in PBS vehicle or vehicle only was injected into the peritoneum. Animals were allowed to recover and were assessed for mesenteric perfusion, mucosal injury, and intestinal cytokines. P values < 0.05 were significant. H2S improved mesenteric perfusion and mucosal injury scores following I/R injury. However, in the setting of eNOS ablation, there was no improvement in these parameters with H2S therapy. Application of H2S also resulted in lower levels of intestinal cytokine production following I/R. Intraperitoneal H2S therapy can improve mesenteric perfusion, intestinal mucosal injury, and intestinal inflammation following I/R. The benefits of H2S appear to be mediated through endothelial nitric oxide-dependent pathways.NEW & NOTEWORTHY H2S is a gaseous mediator that acts as an anti-inflammatory agent contributing to gastrointestinal mucosal defense. It promotes vascular dilation, mucosal repair, and resolution of inflammation following intestinal ischemia and may be exploited as a novel therapeutic agent. It is unclear whether H2S works through nitric oxide-dependent pathways in the intestine. We appreciate that H2S was able to improve postischemic recovery of mesenteric perfusion, mucosal integrity, and inflammation. The beneficial effects of H2S appear to be mediated through endothelial nitric oxide-dependent pathways.

Direct peritoneal resuscitation improves mesenteric perfusion by nitric oxide dependent pathways

BackgroundDirect peritoneal resuscitation (DPR) has been shown to increase survival after intestinal ischemia and reperfusion injury (I/R). We have previously appreciated that minimum essential medium (MEM), a synthetic cell culture medium with bovine serum, glutamine, and antibiotics, contributes to these benefits. We hypothesized that (1) DPR using MEM as a dialysate would increase mesenteric perfusion, improve intestinal mucosal injury, and limit intestinal and hepatic inflammation after intestinal I/R and (2) these improvements would be dependent on endothelial nitric oxide pathways. MethodsEight-week-old C57Bl6J wild-type (WT) and eNOS Knock Out (eNOS KO) male mice were anesthetized and intestinal ischemia was induced for 60 min. After ischemia, 1 mL of phosphate buffered saline vehicle or MEM was injected into the abdominal cavity. Intestinal perfusion was reassessed after 48 h. Animals were then euthanized, and intestines and livers explanted for histologic and molecular analyses. ResultsDPR with MEM significantly improved mesenteric perfusion compared with vehicle (phosphate buffered saline) as measured by Laser Doppler Imaging (WT + MEM 91.58 ± 13.74%, WT + Vehicle 44.27 ± 11.93%, P < 0.05); however, these benefits were lost when endothelial nitric oxide signaling pathways were ablated (eNOS KO + MEM 21.72 ± 5.67 %, eNOS KO + Vehicle 45.24± 11.31%). WT mice treated with MEM also had significantly better preservation of their mucosal architecture (WT + MEM Mdn = 1.0, interquartile range [IQR] = 1.25, WT + Vehicle Mdn = 3.0, IQR = 2.0, P < 0.05). When we compared eNOS KO mice treated with either MEM or vehicle the protective effect of MEM disappeared (eNOS KO + MEM Mdn = 2.0, IQR = 2.25, eNOS KO + Vehicle Mdn = 2.0, IQR = 1.0 P > 0.05). Intestinal levels of interleukin (IL)-1β were increased in WT animals treated with MEM compared with eNOS KOs, whereas concentrations of intestinal IL-6 were similar between groups. Hepatic levels of both IL-1β and IL-6 were significantly elevated in eNOS KOs compared with WT treated with MEM. ConclusionsDPR with MEM has significant therapeutic potential for improving mesenteric perfusion, intestinal injury, and the local inflammatory response after intestinal I/R. These benefits appear to be dependent on nitric oxide signaling within the endothelium.

Nitric oxide is critical for avoiding hepatic lipid overloading via IL-6 induction during liver regeneration after partial hepatectomy in mice.

Nitric oxide (NO), generated from L-arginine by three different isoforms of nitric oxide synthase (NOS), is a pleiotropic factor to regulate physiological functions in almost every organ and tissue. Each knockout mouse of iNOS or eNOS has been used to suggest that NO has a crucial role in liver regeneration after partial hepatectomy (PH), for NO may inhibit caspase 3 activity and is required for EGFR signaling. In previous reports, defective mitochondrial β-oxidation was observed in eNOS KO mice, and hepatic steatosis was often correlated to deficient liver regeneration, so we focused on metabolic perspective and hypothesized that NO depletion in PH mice would affect hepatocytic lipolysis and impair hepatocytes proliferation. We inhibited all NOS isoforms by administrating L-NG-nitroarginine methyl ester (L-NAME) to PH mice, and hepatocyte DNA synthesis was severely inhibited at 40–44 h post PH in L-NAME (+) group. IL-6 was robustly secreted into circulating blood in L-NAME (−) group, but not in L-NAME (+) group. Down-regulation of carnitine palmytoyltransferase 1A, massive lipid accumulation and elevated endoplasmic reticulum (ER) stress relative genes expression level were observed in L-NAME (+) group mouse liver. The expression level of C/EBP homologous protein, a mediator of ER stress induced apoptosis, significantly increased in L-NAME (+) group. Our findings suggest the lack of NO affected IL-6 induction and hepatocyte lipolysis after PH, consequently leading to excessive hepatic lipid accumulation, elevated ER stress and impaired hepatocyte proliferation.

The prosurvival protein BAG3: a new participant in vascular homeostasis.

Bcl2-associated athanogene 3 (BAG3), is constitutively expressed in a few normal cell types, including myocytes, peripheral nerves and in the brain, and is also expressed in certain tumors. To date, the main studies about the role of BAG3 are focused on its pro-survival effect in tumors through various mechanisms that vary according to cellular type. Recently, elevated concentrations of a soluble form of BAG3 were described in patients affected by advanced stage of heart failure (HF), identifying BAG3 as a potentially useful biomarker in monitoring HF progression. Despite the finding of high levels of BAG3 in the sera of HF patients, there are no data on its possible role on the modulation of vascular tone and blood pressure levels. The aim of this study was to investigate the possible hemodynamic effects of BAG3 performing both in vitro and in vivo experiments. Through vascular reactivity studies, we demonstrate that BAG3 is capable of evoking dose-dependent vasorelaxation. Of note, BAG3 exerts its vasorelaxant effect on resistance vessels, typically involved in the blood pressure regulation. Our data further show that the molecular mechanism through which BAG3 exerts this effect is the activation of the PI3K/Akt signalling pathway leading to nitric oxide release by endothelial cells. Finally, we show that in vivo BAG3 administration is capable of regulating blood pressure and that this is dependent on eNOS regulation since this ability is lost in eNOS KO animals.