eNOS Pathway Research Articles

Extremely low frequency electromagnetic stimulation reduces ischemic stroke volume by improving cerebral collateral blood flow

Extremely low frequency electromagnetic stimulation (ELF-EMS) has been considered as a neuroprotective therapy for ischemic stroke based on its capacity to induce nitric oxide (NO) signaling. Here, we examined whether ELF-EMS reduces ischemic stroke volume by stimulating cerebral collateral perfusion. Moreover, the pathway responsible for ELF-EMS-induced NO production was investigated. ELF-EMS diminished infarct growth following experimental stroke in collateral-rich C57BL/6 mice, but not in collateral-scarce BALB/c mice, suggesting that decreased lesion sizes after ELF-EMS results from improved collateral blood flow. In vitro analysis demonstrated that ELF-EMS increased endothelial NO levels by stimulating the Akt-/eNOS pathway. Furthermore, ELF-EMS augmented perfusion in the hind limb of healthy mice, which was mediated by enhanced Akt-/eNOS signaling. In healthy C57BL/6 mouse brains, ELF-EMS treatment increased cerebral blood flow in a NOS-dependent manner, whereas no improvement in cerebrovascular perfusion was observed in collateral-sparse BALB/c mice. In addition, ELF-EMS enhanced cerebral blood flow in both the contra- and ipsilateral hemispheres of C57BL/6 mice subjected to experimental ischemic stroke. In conclusion, we showed that ELF-EMS enhances (cerebro)vascular perfusion by stimulating NO production, indicating that ELF-EMS could be an attractive therapeutic strategy for acute ischemic stroke by improving cerebral collateral blood flow.

Ferulic Acid Alleviates Atherosclerotic Plaques by Inhibiting VSMC Proliferation Through the NO/p21 Signaling pathway

The benefits and risks of inhibiting the proliferation and migration of vascular smooth muscle cells (VSMCs) in atherosclerosis (AS) remain a subject of debate. In this study, we investigated the effect of ferulic acid (FA) on the proliferation and migration of VSMCs induced by platelet-derived growth factor (PDGF) and the associated mechanism and used ApoE-/- mice to study whether the effect of FA on VSMC proliferation and migration is beneficial in alleviating AS plaques. It was found that FA not only reduced blood lipid levels but also promoted the production of nitric oxide (NO) by MOVAS cells through the endothelial nitric oxide synthase (eNOS) pathway, inhibited the migration and proliferation of VSMCs induced by PDGF, promoted the expression of p21 in VSMCs, and exerted a therapeutic effect against AS.

MMP-2 gene silencing attenuates age-dependent carotid stiffness via reduction of elastin degradation and increased eNOS activation

Abstract Background and aims Arterial stiffness is a hallmark of vascular aging. Being characterized by a loss of elasticity of large arterial walls, arterial stiffness is associated with an increased risk of cardiovascular disease (CVD). The age-dependent arterial stiffness is primarily attributed to alterations in the elastic and collagen deposition that is regulated by a number of enzymes, including matrix metalloproteinase-2 (MMP-2). Nevertheless, the mechanistic link between age-dependent arterial stiffness and MMP-2 remains unclear. In this study, we investigated the effect and efficacy of therapeutic MMP-2 knockdown using small interfering RNA (siRNA) on age-dependent arterial stiffness. Methods Pulse wave velocity (PWV) was assessed in the right carotid artery of wild-type (WT) mice of different age groups. MMP-2 levels and activity in the carotid artery and plasma of young (3 months) and aged (20–25 months) WT mice were determined. Old WT mice (18–21 months) were treated for 4 weeks with either MMP-2 or scrambled siRNA, in which carotid PWV was assessed at baseline, 2 and 4 weeks after the start of the treatment. Elastin to collagen ratio, desmosin (DES) level, and endothelial nitric oxide synthase (eNOS) pathways were also evaluated and compared. Lastly, levels of circulating MMP-2 and DES, the breakdown product of elastin, were measured in a human cohort (23–86 years old), in whom carotid-femoral PWV was assessed. Results Carotid PWV, as well as both vascular and circulating MMP-2 levels, were elevated with increasing age in WT mice (Figure 1). Therapeutic MMP-2 knockdown in aged WT mice reduced the vascular MMP-2 expression and attenuated age-dependent carotid stiffness. Increased elastin to collagen ratio and a lower plasma DES level were observed on MMP-2 silenced treated animals (Figure 2). Moreover, siMMP-2 treated mice showed enhanced eNOS phosphorylation on Ser1177. A direct interaction between MMP-2 and eNOS was also observed, which, interestingly, is augmented with age. Finally, collected human data showed a higher level of circulating MMP-2 levels on the elderly subjects. In addition, plasma DES level is positively correlated with age and aortic PWV, indicating the involvement of vascular elastin catabolism on arterial stiffness. Conclusions Therapeutic MMP-2 gene silencing, specifically targeting vascular MMP-2, attenuates age-dependent carotid stiffness. This effect is mediated by augmenting eNOS activation and reducing elastin degradation. Thus, our findings indicate MMP-2 as a potential therapeutic target to mitigate age-dependent arterial stiffness and CVD. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swiss National Science Foundation,Foundation for Cardiovascular Research–Zurich Heart House Figure 1

Knockdown of miR-423-5p simultaneously upgrades the eNOS and VEGFa pathways in ADSCs and improves erectile function in diabetic rats.

This study aimed to explore the possibility of miR‐423‐5p modified adipose‐derived stem cell (ADSCs) therapy on streptozotocin (STZ)‐induced diabetes mellitus erectile dysfunction (DMED) rats. MiR‐423‐5p was knocked down in ADSCs. ADSCs, NC‐miR‐ADSCs and miR‐ADSCs were co‐cultured with human umbilical vein endothelial cells (HUVECs). Normal and high glucose media were supplemented. The supernatant and HUVECs were collected for assessment of eNOS and VEGFa expression, cell proliferation, and apoptosis. HUVECs co‐cultured with ADSCs or miR‐ADSCs exhibited higher eNOS and VEGFa protein expression levels compared to DM groups. MiR‐ADSCs enhanced HUVEC proliferation compared to the ADSCs and NC‐miR‐ADSCs. Lower apoptotic rates were observed when HUVECs were co‐cultured with miR‐ADSCs, compared to ADSCs and NC‐miR‐ADSCs. Fifteen male Sprague‐Dawley (SD) rats aged 12 weeks were induced to develop diabetes mellitus by intraperitoneal injection with STZ, and five healthy SD rats were used as normal controls. Eight weeks after developing diabetes, the rats received ADSCs and miR‐ADSCs via injection into the corpora cavernosa, whereas normal controls and DM controls were injected with saline. Erectile function and histological assessment of penile tissues were performed 8 weeks after injection. The ICP/MAP indicated that erectile function was impaired in the DM rats compared with the normal group. Injection of ADSCs and miR‐ADSCs improved erectile function significantly and was associated with the overexpression of eNOS and VEGFa. MiR‐423‐5p knockdown in ADSCs ameliorated high glucose‐mediated damage to HUVECs and improved erectile function in DM rats by inducing eNOS and VEGFa overexpression, indicating that miR‐423‐5p may be a potential target in the treatment of DMED.

Ex vivo Akt inhibition reverses castration induced internal pudendal artery and penile endothelial dysfunction

AimsAndrogen deprivation therapy is a common prostate cancer treatment which causes men to have castrate levels of testosterone. Unfortunately, most testosterone deficient patients will suffer severe erectile dysfunction (ED) and have no effective ED treatment options. Testosterone deficiency causes endothelial dysfunction and impairs penile vasodilation necessary to maintain an erection. Recent evidence demonstrates testosterone activates androgen receptors (AR) and generates nitric oxide (NO) through the Akt-endothelial NO synthase (eNOS) pathway; however, it remains unknown how castration impacts this signaling pathway. Materials and methodsIn this study, we used a surgically castrated rat model to determine how castration impacts ex vivo internal pudendal artery (IPA) and penile relaxation through the Akt-eNOS pathway. Key findingsUnlike systemic vasculature, castration causes significant IPA and penis endothelial dysfunction associated with a 50% AR reduction. Though testosterone and acetylcholine (ACh) both phosphorylate Akt and eNOS, castration did not affect testosterone-mediated IPA and penile Akt or eNOS phosphorylation. Surprisingly, castration increases ACh-mediated Akt and eNOS phosphorylation but reduces the eNOS dimer to monomer ratio. Akt inhibition using 10DEBC preserves IPA eNOS dimers. Functionally, 10DEBC reverses castration induced ex vivo IPA and penile endothelial dysfunction. SignificanceThese data demonstrate how castration uncouples eNOS and provide a novel strategy for improving endothelial-dependent relaxation necessary for an erection. Further studies are needed to determine if Akt inhibition may treat or even prevent ED in testosterone deficient prostate cancer survivors.

Pravastatin attenuates sepsis-induced acute lung injury through decreasing pulmonary microvascular permeability via inhibition of Cav-1/eNOS pathway

BackgroundDisruption of alveolar endothelial barrier caused by inflammation drives the progression of septic acute lung injury (ALI). Pravastatin, an inhibitor of HMG Co-A reductase, has potent anti-inflammatory effects. In the present study, we aim to explore the beneficial role of pravastatin in sepsis‐induced ALI and its related mechanisms. MethodsA septic ALI model was established by cecal ligation and puncture (CLP) in mice. The pulmonary microvascular endothelial cells (PMVECs) were challenged with lipopolysaccharide (LPS). The pathological changes in lung tissues were examined by HE staining. The pulmonary microvascular permeability was determined by lung wet-to-dry (W/D) weight ratio and Evans blue staining. The total protein concentration in bronchoalveolar lavage fluid (BALF) was detected by BCA assay. The levels of TNF-α, IL-1β, and IL-6 were assessed by qRT-PCR and ELISA. Apoptosis was determined by flow cytometry and TUNEL. Western blotting was performed for detection of target protein levels. The expression of VE-Cadherin in lung tissues was evaluated by immunohistochemical staining. ResultsPravastatin improved survival rate, attenuated lung pathological changes and reduced pulmonary microvascular permeability in septic mice. In addition, pravastatin restrained sepsis-induced inflammatory response and apoptosis in the lung tissues and PMVECs. Moreover, pravastatin up-regulated the levels of junction proteins ZO-1, JAM-C, and VE-Cadherin. Finally, pravastatin suppressed inflammation, apoptosis and enhanced the expression of junction proteins via regulating Cav-1/eNOS signaling pathway in LPS-exposed PMVECs. ConclusionPravastatin ameliorates sepsis-induced ALI through improving alveolar endothelial barrier disruption via modulating Cav-1/eNOS pathway, which may be an effective candidate for treating septic ALI.

A xanthene derivative, DS20060511, attenuates glucose intolerance by inducing skeletal muscle-specific GLUT4 translocation in mice

Reduced glucose uptake into the skeletal muscle is an important pathophysiological abnormality in type 2 diabetes, and is caused by impaired translocation of glucose transporter 4 (GLUT4) to the skeletal muscle cell surface. Here, we show a xanthene derivative, DS20060511, induces GLUT4 translocation to the skeletal muscle cell surface, thereby stimulating glucose uptake into the tissue. DS20060511 induced GLUT4 translocation and stimulated glucose uptake into differentiated L6-myotubes and into the skeletal muscles in mice. These effects were completely abolished in GLUT4 knockout mice. Induction of GLUT4 translocation by DS20060511 was independent of the insulin signaling pathways including IRS1-Akt-AS160 phosphorylation and IRS1-Rac1-actin polymerization, eNOS pathway, and AMPK pathway. Acute and chronic DS20060511 treatment attenuated the glucose intolerance in obese diabetic mice. Taken together, DS20060511 acts as a skeletal muscle-specific GLUT4 translocation enhancer to facilitate glucose uptake. Further studies of DS20060511 may pave the way for the development of novel antidiabetic medicines.

Peroxidasin promotes diabetic vascular endothelial dysfunction induced by advanced glycation end products via NOX2/HOCl/Akt/eNOS pathway

Reactive oxygen species (ROS) derived from NADPH oxidases (NOX) plays an essential role in advanced glycation end products (AGEs)-induced diabetic vascular endothelial dysfunction. Peroxidasin (PXDN, VPO1) is one member of peroxidases family that catalyzes hydrogen peroxide (H2O2) to hypochlorous acid (HOCl). This present study aimed to elucidate the role of PXDN in promoting vascular endothelial dysfunction induced by AGEs in diabetes mellitus. We found that, compared to non-diabetic (db/m) mice, PXDN expression was notably increased in db/db mice with impaired endothelium-dependent relaxation. Knockdown of PXDN in vivo through tail vein injection of siRNA restored the impaired endothelium-dependent relaxation function of db/db mice which is accompanied with up-regulation of eNOS Ser1177 phosphorylation and NO production. AGEs significantly elevated expression of PXDN and 3-Cl-Tyr, but decreased phosphorylation of Akt and eNOS and NO release in HUVECs. All these effects induced by AGEs were remarkable alleviated by silencing PXDN with small interfering RNAs. In addition, HOCl treatment alone as well as HOCl added with Akt inhibitor MK2206 inhibited phosphorylation of Akt and eNOS, reducing NO production. More importantly,AGEs-induced up-regulation of PXDN and 3-Cl-Tyr with endothelial dysfunction were transformed by NOX2 silencing and H2O2 scavengers. Thus, these results support the conclusion that PXDN promotes AGEs-induced diabetic vascular endothelial dysfunction by attenuating eNOS phosphorylation at Ser1177 via NOX2/HOCl/Akt pathway.

Estrogen and Preeclampsia: Potential of Estrogens as Therapeutic Agents in Preeclampsia.

There is a significant decline in the estrogen levels in preeclampsia, and exogenous administration of estradiol normalizes blood pressure and other associated symptoms of preeclampsia. The decrease in estrogen levels may be due to changes in enzyme activities of hydroxysteroid (17-β) dehydrogenase 1, aromatase, and COMT. There is also a decrease in the novel, estrogenic G-protein-coupled receptor 30 (GPR30) in the placental trophoblast cells in preeclampsia. The activation of GPR30 protects the placenta from hypoxia-reoxygenation injury, decreases apoptosis and increases proliferation through eNOS and PI3K-Akt signaling pathways. Estrogens may also increase Ca2+-activated K+ channel function, decrease the release of inflammatory cytokines, and oxidative stress to improve placental perfusion. Both preclinical and clinical studies show the decrease in the 2-methoxyestradiol levels in preeclampsia, which may be due to a decrease in estradiol itself along with a decrease in the enzymatic actions of the COMT enzyme. 2-Methoxyestradiol activates HIF1α and vascular endothelial growth factor receptors (VEGFR-2) to maintain placental perfusion by increasing angiogenesis. The present review discusses the preclinical and clinical studies describing the role of estrogen in preeclampsia along with possible mechanisms.

Effects of sitagliptin on ß-adrenoceptor mediated relaxation in streptozotocin-diabetic rat aorta

Background/aim Dipeptidyl peptidase-4 (DPP4) inhibitors, a class of oral antidiabetic drugs, have been shown to be protective on the vascular system because of their antiinflammatory, antiatherosclerotic, and vasodilatory effects. ß 2-adrenoceptors (ß 2-ARs) mediate the vasorelaxation in the aorta. However, ß 3-adrenoceptor-mediated relaxation has not been studied in diabetic aorta yet. Thus, we aimed to study the effect of sitagliptin treatment on ß 2- and ß3-adrenoceptor-mediated relaxations in the diabetic rat aorta.Materials and methods Eight-week old Sprague Dawley rats were divided into three groups: control, diabetic, sitagliptin treated diabetic. Diabetes was induced by injection of streptozotocin (35 or 40 mg/kg, intraperitoneally). After 10 weeks of diabetes, some of the diabetic rats were treated with sitagliptin (orally, 10mg/kg/day). ß2- and ß 3-AR-mediated relaxation responses were evaluated by using isoprenaline and CL 316,243, respectively. ß3-AR-mediated relaxation experiments were repeated in presence of L-NAME. Western blotting and immunohistochemistry were performed to determine the abundance of ß3-adrenoceptor and endothelial nitric oxide synthase (eNOS).Results The isoprenaline-mediated relaxation response was impaired in the diabetic group and sitagliptin treatment did not improve it. There was no significant change in CL316,243 mediated-relaxation or protein expression of ß 3-ARs among the groups. However, the ratio of phosphorylated eNOS/NOS protein was increased markedly in the sitagliptin treated group, which points the stimulating effect of this drug towards the eNOS pathway. Conclusion Our results indicate that sitagliptin treatment does not alter ß- AR-mediated relaxation in streptozotocin-diabetic rat aorta; however, it significantly stimulates the eNOS pathway. Future studies are needed to clarify the relationship between the eNOS pathway and DPP-4 inhibition.

Tadalafil ameliorates bladder overactivity by restoring insulin-activated detrusor relaxation via the bladder mucosal IRS/PI3K/AKT/eNOS pathway in fructose-fed rats

The pathophysiologies of metabolic syndrome (MS) and overactive bladder (OAB) might overlap. Using fructose-fed rats (FFRs) as a rodent model of MS we investigated the effects of tadalafil (a phosphodiesterase type 5 inhibitor) on the dysregulated insulin signalling in the bladder mucosa and bladder overactivity. Micturition behaviour was evaluated. Concentration–response curves on detrusor relaxation to insulin stimulation were examined. Expression and phosphorylation of proteins in the insulin signalling pathway were evaluated by Western blotting. Levels of detrusor cGMP and urinary nitrite and nitrate (NOx) were measured. We observed FFRs exhibited metabolic traits of MS, bladder overactivity, and impaired insulin-activated detrusor relaxation in organ bath study. A high-fructose diet also impeded insulin signalling, reflected by overexpression of IRS1/pIRS1Ser307 and pIRS2Ser731 and downregulation of PI3K/pPI3KTyr508, AKT/pAKTSer473, and eNOS/peNOSSer1177 in the bladder mucosa, alongside decreased urinary NOx and detrusor cGMP levels. Tadalafil treatment restored the reduced level of mucosal peNOS, urinary NOx, and detrusor cGMP, improved the insulin-activated detrusor relaxation, and ameliorated bladder overactivity in FFRs. These results suggest tadalafil may ameliorate MS-associated bladder overactivity by restoring insulin-activated detrusor relaxation via molecular mechanisms that are associated with preservation of IR/IRS/PI3K/AKT/eNOS pathway in the bladder mucosa and cGMP production in the bladder detrusor.

Chronic nitrite treatment activates adenosine monophosphate-activated protein kinase-endothelial nitric oxide synthase pathway in human aortic endothelial cells

• Chronic nitrite treatment increases phosphorylation level of AMPK at Thr172 under physiological concentrations. • Chronic nitrite treatment increases phosphorylation level of eNOS at Ser1177 under physiological concentrations. • Nitrite may be an activator of the AMPK–eNOS pathway in human aortic endothelial cells. • Nitrite may increase NO production by activating AMPK, followed by eNOS activation in addition to non-enzymatic reduction. Endothelial dysfunction, with impaired bioavailability and/or bioactivity of the vasoprotective molecule, nitric oxide, appears to be a vital step in the initiation of atherosclerosis. Several studies have shown that dietary nitrate/nitrite can have significant benefits on human cardiovascular homeostasis. Although serum nitrite concentrations can reach micromolar levels, the physiological significance of nitrate/nitrite in normal tissues has not been fully elucidated. We investigated in vitro the chronic effects of nitrate/nitrite on endothelial nitric oxide synthase (eNOS) to determine the potential vasoprotective effects of nitrate/nitrite and the underlying molecular mechanisms. Our results demonstrate the expression of phosphorylated eNOS at Ser1177 and phosphorylated adenosine monophosphate activated protein kinase (AMPK) at Thr172 in human aortic endothelial cells were increased after nitrite treatment. We suggest that nitrite stimulation may enhance eNOS activation, which is due, in part, to AMPK activation. The AMPK–eNOS activation by nitrite may be a possible molecular mechanism underlying the vascular protective effects of dietary nitrate.

Endothelial specific deletion of HMGB1 increases blood pressure and retards ischemia recovery through eNOS and ROS pathway in mice

Recent studies demonstrated HMGB1, an extracellular inflammation molecule, played an important role on endothelial cells. This study aimed to define the role and related mechanism of HMGB1 in endothelial cells. Endothelial-specific deletion of HMGB1(HMGB1ECKO) was generated and Akt/eNOS signaling, reactive oxygen species (ROS) production, endothelium dependent relaxation (EDR), and angiogenesis were determined in vitro and in vivo. Decreased activation of Akt/eNOS signaling, sprouting, and proliferation, and increased ROS production were evidenced in endothelial cells derived from HMGB1ECKO mice as compared with wild type controls. Decreased EDR and retarded blood flow recovery after hind limb ischemia were also demonstrated in HMGB1ECKO mice. Both impaired EDR and angiogenesis could be partly rescued by superoxide dismutase in HMGB1ECKO mice. In conclusion, intracellular HMGB1 might be a key regulator of endothelial Akt/eNOS pathway and ROS production, thus plays an important role in EDR regulation and angiogenesis.

Fenofibrate attenuates doxorubicin-induced cardiac dysfunction in mice via activating the eNOS/EPC pathway

Endothelial progenitor cells (EPCs) improve endothelial impairment, which in turn restores endothelial function in patients with heart failure (HF). In the present study, we tested whether fenofibrate, with its anti-inflammatory and vasoprotective effects, could improve myocardial function by activating EPCs through the eNOS pathway in a doxorubicin (DOX)-induced cardiomyopathy mouse model. Wild-type mice were divided into 4 groups and treated with vehicle, DOX + saline, DOX + fenofibrate, and DOX + fenofibrate + L-NAME (N(ω)-nitro-L-arginine methyl ester). DOX-induced cardiac atrophy, myocardial dysfunction, the number of circulating EPCs and tissue inflammation were analyzed. Mice in the DOX + fenofibrate group had more circulating EPCs than those in the DOX + saline group (2% versus 0.5% of total events, respectively) after 4 weeks of treatment with fenofibrate. In addition, the inhibition of eNOS by L-NAME in vivo further abolished the fenofibrate-induced suppression of DOX-induced cardiotoxic effects. Protein assays revealed that, after DOX treatment, the differential expression of MMP-2 (matrix metalloproteinase-2), MMP-9 (matrix metalloproteinase-9), TNF-α (tumor necrosis factor-α), and NT-pro-BNP (N-terminal pro-B-type natriuretic peptide) between saline- and DOX-treated mice was involved in the progression of HF. Mechanistically, fenofibrate promotes Akt/eNOS and VEGF (vascular endothelial growth factor), which results in the activation of EPC pathways, thereby ameliorating DOX-induced cardiac toxicity.

PARP inhibitor Veliparib (ABT-888) enhances the anti-angiogenic potentiality of Curcumin through deregulation of NECTIN-4 in oral cancer: Role of nitric oxide (NO)

Concurrent use of DNA damaging agents with PARP inhibitors contribute to the effectiveness of the anticancer therapy. But there is a dearth of reports on the antiangiogenic effects of PARP inhibitors and the suppression of angiogenesis by this drug combination is not yet reported. For the successful development of cancer therapeutics, anti-cancer drugs ought to have anti-angiogenic potentiality along with their DNA damaging abilities. In this current piece of work, we investigated the in vitro and in ovo anti-angiogenic effect of Curcumin and Veliparib (a PARP inhibitor) in oral cancer. Recent evidences suggest an involvement of the NECTIN-4 in cancer angiogenesis and the exact molecular pathway of this involvement remains to be delineated. We observed that the soluble NECTIN-4 secreted from H357 oral cancer cells enhanced the angiogenesis of endothelial cells (HUVECs) and this was inhibited by Curcumin-Veliparib combination. NECTIN-4 enhanced vascularization, induced vasodilation and triggered the angiogenic sprouting via endothelial tip cell filopodia. Data indicated that NECTIN-4 mediated angiogenesis is associated with PI3K-AKT-mediated nitric oxide (NO) formation. A noticeable increase in the NO enhanced epithelial NO level through HIF-1α mediated iNOS activation. We observed that increased NO enhanced the NECTIN-4 mediated eNOS expression and thereby elicited further angiogenesis. Curcumin antagonised the NECTIN-4-induced angiogenesis through inhibition of PI3K-AKT mediated eNOS pathway and Veliparib synergized the effect of Curcumin. Our observations indicate that NO is cardinal in inducing NECTIN-4 mediated angiogenesis in H357 cells. Thus, Curcumin-Veliparib combination suppresses angiogenesis through deregulation of the PI3K-AKT-eNOS pathway downstream to the NECTIN-4.

High phosphate impairs arterial endothelial function through AMPK-related pathways in mouse resistance arteries.

In patients with renal disease, high serum phosphate shows a relationship with cardiovascular risk. We speculate that high phosphate (HP) impairs arterial vasodilation via the endothelium and explore potential underlying mechanisms. Isolated vessel relaxation, endothelial function, glomerular filtration rate (GFR), oxidative stress status and protein expression were assessed in HP diet mice. Mitochondrial function and protein expression were assessed in HP-treated human umbilical vein endothelial cells (HUVECs). High phosphate (1.3%) diet for 12weeks impaired endothelium-dependent relaxation in mesenteric arteries, kidney interlobar arteries and afferent arterioles; reduced GFR and the blood pressure responses to acute administration of acetylcholine. The PPARα/LKB1/AMPK/eNOS pathway was attenuated in the endothelium of mesenteric arteries from HP diet mice. The observed vasodilatory impairment of mesenteric arteries was ameliorated by PPARα agonist WY-14643. The phosphate transporter PiT-1 knockdown prevented HP-mediated suppression of eNOS activity by impeding phosphorus influx in HUVECs. Endothelium cytoplasmic and mitochondrial reactive oxygen species (ROS) were increased in HP diet mice. Moreover HP decreased the expression of mitochondrial-related antioxidant genes. Finally, mitochondrial membrane potential and PGC-1α expression were reduced by HP treatment in HUVECs, which was partly restored by AMPKα agonist. HP impairs endothelial function by reducing NO bioavailability via decreasing eNOS activity and increasing mitochondrial ROS, in which the AMPK-related signalling pathways may play a key role.

Abstract 13295: Novel Experimental Therapeutics for COVID-19 Derived From a Nitric Oxide Donor

Urea transport protein B, the product of the gene SLC14A1 , facilitates transport of urea, water and urea analogues across cell membranes. SLC14A1 mRNA is overexpressed in human vascular endothelial cells in culture under hypoxic (1% oxygen) conditions compared with normoxia. This leads to transport of urea out of the endothelial cell and likely contributes to the reduction in eNOS (endothelial nitric oxide synthase) pathway activity in hypoxia. NO has antiviral activity. Novel compounds were developed by binding a urea-like moiety to the backbone of the generic agent isosorbide mononitrate, a well-known NO donor, to combat vascular endothelial dysfunction in COVID-19, a disease characterized by systemic hypoxia and inflammation due to SARS-CoV-2 infection. A study of drug-protein interactions was undertaken using in silico modelling. Novel compounds were studied against 9 key SARS-CoV-2 targets using Maestro, Schrödinger Suite software (Glide docking). Docking scores and intermolecular interactions within the target’s key binding amino acid residues were studied to compare investigational compounds and known antivirals. Several novel agents tested had a better Glide Score (a prediction of ligand affinity) against the papain-like protease (PL pro ) of SARS-CoV-2 compared with known antiviral drugs. PL pro is considered to be a primary target for therapeutic inhibition of the SARS viruses. The candidate compounds CR-305, CR-607, CR-510 and CR-605 were all superior to Remdesivir, GS-441524, Lopinavir, Boceprevir, and Ribavirin. Given the known direct antiviral action of NO and evidence of specific binding of these compounds to the PL pro of SARS-CoV-2 based on the in silico results, we conclude there is a high likelihood these novel compounds will prove to be of therapeutic value against COVID-19. CR-305 appears to have a higher affinity to SARS-CoV-2 than other antivirals as it sits firmly in the PL pro catalytic pocket and makes the most of key interactions with the catalytic pocket residues: Gly163, Asp164, Gln271 and Tyr264. These data call for a new focus on these novel antiviral agents as they appear to bind with an increased avidity to PL pro (compared with other known antivirals) while targeting delivery of NO to the SARS-CoV-2 virus in COVID-19.

Titanate nanofibers reduce Kruppel-like factor 2 (KLF2)-eNOS pathway in endothelial monolayer: A transcriptomic study

Although titanate nanofibers (TiNFs) and titanate nanotubes (TiNTs) have been proposed as relatively biocompatible nanomaterials (NMs), there is currently lacking of systemic studies which investigated the toxicity of TiNFs and TiNTs to endothelium. In this study, we developed endothelial monolayer model by using cell culture inserts, and systemically investigated the toxicity of TiNFs and TiNTs by RNA-seq, with a focus on Kruppel-like factor (KLF)-mediated effects, since KLF are transcription factors (TF) involved in the regulation of vascular biology. It was shown that NMs did not significantly induce cytotoxicity despite substantial internalization. However, the expression of many KLF was altered, and Western blot further confirmed that NMs down-regulated KLF2 proteins. Ingenuity pathway analysis (IPA) revealed that NMs altered the expression of KLF2-targed genes, typically the genes involved in inflammatory responses. KLF2-related Gene Ontology (GO) terms and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways were also altered, and it should be noticed that NMs altered GO terms and KEGG pathways related with endothelial NO synthase (eNOS). This study further verified that NMs decreased intracellular NO and eNOS proteins. All the observed effects were more obvious for TiNFs compared with TiNTs. Combined, this study showed that TiNFs or TiNTs were non-cytotoxic to endothelial monolayer model, but TiNFs and more modestly TiNTs decreased KLF2 leading to decreased eNOS proteins and NO production. Our data may provide novel understanding about the toxicity of TiNFs as well as other Ti-based NMs to endothelium.

Blocking exosomal miRNA-153-3p derived from bone marrow mesenchymal stem cells ameliorates hypoxia-induced myocardial and microvascular damage by targeting the ANGPT1-mediated VEGF/PI3k/Akt/eNOS pathway

It has been widely reported that exosomes derived from mesenchymal stem cells (MSCs) have a protective effect on myocardial infarction (MI). However, the specific molecules which play a damaging role in MSCs shuttled miRNAs are much less explored. MiRNA-153-3p (miR-153-3p) is a vital miRNA which has been proved to modulate cell proliferation, apoptosis, angiogenesis, peritoneal fibrosis and aortic calcification. Here, we aim to study the effect and mechanism of miR-153-3p in MSC-derived exosomes on hypoxia-induced myocardial and microvascular damage. The exosomes of MSCs were isolated and identified, and the MSCs-exosomes with low expression of miR-153-3p (exo-miR-153-3p−) were constructed to interfere with the endothelial cells and cardiomyocytes in the oxygen-glucose deprivation (OGD) model. The viability, apoptosis, angiogenesis of endothelial cells and cardiomyocytes were determined. Additionally, ANGPT1/VEGF/VEGFR2/PI3K/Akt/eNOS pathway was detected by ELISA and/or western blot. The results illustrated that exo-miR-153-3p− significantly reduced the apoptosis of endothelial cells and cardiomyocytes and promoted their viability. Meanwhile, exo-miR-153-3p− can promote the angiogenesis of endothelial cells. Mechanistically, miR-153-3p regulates the VEGF/VEGFR2/PI3K/Akt/eNOS pathways by targeting ANGPT1. Intervention with VEGFR2 inhibitor (SU1498, 1 μM) remarkably reversed the protective effect of exo-miR-153-3p− in vascular endothelial cells and cardiomyocytes treated by OGD. Collectively, MSCs-derived exosomes with low-expressed miR-153-3p notably promotes the activation of ANGPT1 and the VEGF/VEGFR2 /PI3K/Akt/eNOS pathways, thereby preventing the damages endothelial cells and cardiomyocytes against hypoxia.

Upregulation of the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas receptor axis by a combination of Yinyanghuo (Herba Epimedii Brevicornus) and Cheqianzi (Semen Plantaginis) improves erectile function in spontaneously hypertensive rats.

To evaluate the effects of a combination of Yinyanghuo (Herba Epimedii Brevicornus) (HEB) and Cheqianzi (Semen Plantaginis) (SP) on erectile dysfunction caused by essential hypertension in spontaneously hypertensive rats (SHRs), and to elucidate the role of the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas receptor (ACE2/Ang [1-7]/Mas receptor) axis in this process. A total of 24 SHRs were randomly assigned to three groups: SHR-control, low-dose (12.5 g/kg) and high-dose (25 g/kg) HEB+SP (HEBSP). Eight Wistar-Kyoto rats were used as normal controls. HEBSP was administered by oral gavage for 28 d. Erectile function was measured once a week using the Heaton test. After 4 weeks of treatment, the corpus cavernosum was harvested from each rat to measure nitric oxide (NO), nitric oxide synthase (eNOS) and Ang (1-7) levels, as well as ACE2, Mas receptor and neuronal nitric oxide synthase (nNOS) protein expression. After 4 weeks of treatment, HEBSP significantly increased erectile function in the treated group compared with SHR-control group (P < 0.01). Additionally, HEBSP treatment significantly increased cavernosal levels of Ang (1-7), eNOS and NO. Moreover, HEBSP significantly elevated the expression levels of ACE2, Mas receptor and nNOS. These beneficial effects were elevated in the high-dose HEBSP group. HEBSP improved erectile function in SHRs by upregulating the ACE2/Ang (1-7)/Mas receptor axis, eNOS and nNOS pathways.