Endothelial Cell Nitric Oxide Synthase Research Articles

Novel Role of 5-Methyl-(6S)-Tetrahydrofolate in Mediating Endothelial Cell Tetrahydrobiopterin in Pregnancy and Implications for Gestational Hypertension.

Folate intake during pregnancy is essential for fetal development and maternal health. However, the specific effects of folic acid (FA) and 5-methyl-(6S)-tetrahydrofolate (5-MTHF) on the prevention and treatment of hypertensive disorders of pregnancy remain unclear. We investigated whether FA and 5-MTHF have different effects on endothelial cell tetrahydrobiopterin (BH4) metabolism in pregnancy and the possible consequences for endothelial NO generation, maternal blood pressure, and fetal growth. We analyzed the maternal blood pressure in pregnant wild-type (Gch1fl/fl) and Gch1fl/fl Tie2cre mice treated with either FA or 5-MTHF starting before pregnancy, mid-pregnancy or late pregnancy. BH4, superoxide, and NO bioavailability were determined in mouse and human models of endothelial cell BH4 deficiency by high-performance liquid chromatography. In vitro studies in mouse and human endothelial cells showed that treatment with 5-MTHF, but not FA, elevated BH4 levels, reduced superoxide production, and increased NO synthase activity. In primary endothelial cells isolated from women with hypertensive pregnancies, exposure to 5-MTHF, but not FA, restored the reduction in BH4 levels and NO synthase activity. In vivo studies in mice revealed that oral treatment with 5-MTHF, but not FA, prevented and treated hypertension in pregnancy when administered either before or during pregnancy, respectively, and normalized placental and fetal growth restriction if administered from mid-gestation onward. Collectively, these studies identify a critical role for 5-MTHF in endothelial cell function in pregnancy, related to endothelial cell BH4 availability and NO synthase activity. Thus, 5-MTHF represents a novel therapeutic agent that may potentially improve endothelial function in hypertensive disorders of pregnancy by targeting endothelial cell BH4.

Protective effects of engineered Lactobacillus crispatus strains expressing G-CSF on thin endometrium of mice.

Does recombinant Lactobacillus expressing granulocyte colony-stimulating factor (G-CSF) have a better protective effect than the current treatment of thin endometrium (TE)? This study suggested that the intrauterine injection of Lactobacillus crispastus (L. crispastus)-pPG612-G-CSF has a positive effect on preventing TE induced by 95% alcohol in mice. TE has a negative impact on the success rate of ART in patients, and is usually caused by intrauterine surgery, endometrial infection, or hormone drugs. Exogenous G-CSF can promote endometrial vascular remodelling and increase endometrial receptivity and the embryo implantation rate. Moreover, Lactobacillus plays a crucial role in maintaining and regulating the local microecological balance of the reproductive tract, and it could be a delivery carrier of the endometrial repair drug G-CSF. We constructed engineered L. crispastus strains expressing G-CSF. The mice were divided into five groups: (i) Control group (C, n = 28), uteri were treated with preheated saline solution via intrauterine injection on the third and sixth day of oestrus; (ii) Model group (M, n = 35), where uteri were treated with 95% alcohol on the third day of oestrus and preheated saline solution on the sixth day of oestrus via intrauterine injection; (iii) L. crispatus-pPG612-treatment group (L, n = 45), where uteri were treated with 95% alcohol on the third day of oestrus and 0.1 ml × 108 CFU/ml L. crispatus-pPG612 on the sixth day of oestrus via intrauterine injection; (iv) L. crispatus-pPG612-treatment group (LG, n = 45), where uteri were treated with 95% alcohol on the third day of oestrus and 0.1 ml × 108 CFU/ml L. crispatus-pPG612-G-CSF on the sixth day of oestrus via intrauterine injection; (v) G-CSF-treatment group (G, n = 52), where uteri were treated with 95% alcohol on the third day of oestrus and 30 µg/kg G-CSF on the sixth day of oestrus via intrauterine injection. Then, we compared the effects of L. crispastus, L. crispatus-pPG612-G-CSF and G-CSF on endometrial thickness, angiogenesis, fibrosis, and inflammation in the TE mouse. We collected uterine tissues for haematoxylin-eosin staining, immunohistochemical staining, Western blot and RT-PCR, as well as serum for ELISA and uterine flushing solution for high-throughput sequencing. Compared with those in the M group (the mice of the group were intrauterine injected 95% alcohol and treated with saline solution), the L. crispatus-pPG612-G-CSF strain increased the thickness of the endometrium (P < 0.001) and the number of blood vessels and glands (both P < 0.001), enhanced the expression of cytokeratin 19 (CK19) (P < 0.001), vimentin (Vim) (P < 0.001), vascular endothelial growth factor-A (P < 0.001), and CD34 (P < 0.001), and decreased fibrosis levels (P = 0.004). In addition, the high-throughput sequencing results indicated that the L. crispatus-pPG612-G-CSF strain could decrease the abundance of Pseudomonas (P = 0.044) and Actinomyces spp. (P = 0.094) in TE mice and increased the average number of embryos (P = 0.036). Finally, the L. crispatus-pPG612-G-CSF strain was preliminarily confirmed to activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signalling pathway and enhance the mRNA expression of hypoxia-inducible factor-1α (P < 0.001), vascular endometrial growth factor (P = 0.003), and endothelial cell nitric oxide synthase (P = 0.003) in mouse uterine tissue. N/A. Therapy with the L. crispatus-pPG612-G-CSF strain has tremendous potential to accelerate the reparative processes of TE. However, we have reported only the expression of genes and proteins related to the PI3K/AKT pathway, and numerous other mechanisms may also be involved in the restoration of the endometrium by L. crispatus-pPG612-G-CSF. The results from the study provide new ideas and suggest new methods for TE treatment. This work was financially supported by the Project of Science and Technology Development Plan of Jilin Province (grant number 20210101232JC), the Science and Technology Plan Item of Jilin Provincial Education Department (grant number JT53101022010), and the Doctoral Research Start-up Fund of Jilin Medical University (grant numbers JYBS2021014LK and 2022JYBS006KJ). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

The proteins derived from platelet-rich plasma improve the endothelialization and vascularization of small diameter vascular grafts

Vascular transplantation has become an ideal substitute for heart and peripheral vascular bypass therapy and tissue-engineered vascular grafts (TEVGs) present an attractive potential solution for vascular surgery. However, small diameter (Ф < 6 mm) vascular do not have ideal TEVGs for clinical use. Platelet-rich plasma (PRP), a key source of bioactive molecules, has been confirmed to promote tissue repair and regeneration. In this study, we prepared PRP-loaded TEVGs (PRP-TEVGs) by electrospinning, investigated the characterization of TEVGs, and verified the effect of PRP-TEVGs in vivo and in vitro experiments. The results suggested that PRP-TEVGs had good biocompatibility, released growth factors stably, promoted cell proliferation and migration significantly, up-regulated the expression of endothelial NO synthase (eNOS) in functional vascular endothelial cells (VECs), and maintained the stability of the endothelial structure. In vivo experiments suggest that PRP can promote rapid endothelialization and reconstruction of TEVGs. Overall, this finding indicated that PRP could promote the rapid vascular endothelialization of small-diameter TEVGs by improving contractile vascular smooth muscle cells (VSMCs) regeneration, and maintaining the integrity and functionality of VECs.

Lycium barbarum polysaccharide antagonizes cardiomyocyte apoptosis by inhibiting the upregulation of GRK2 induced by I/R injury, and salvage mitochondrial fission/fusion imbalance and AKT/eNOS signaling

Ischemia–reperfusion (I/R) injury is the main reason why infarct size continues to progress during the process of restoring myocardial perfusion, and it significantly increases the risk of death. At present, the therapeutic effects of clinically used drugs are limited. Therefore, it is particularly necessary to explore myocardial-protective agents that effectively prevent I/R injury. Lycium barbarum polysaccharide (LBP) is a water-soluble polysaccharide extracted from wolfberry fruit. In this study, we found that LBP limited myocardial infarct size, improved adverse remodeling, and reduced cell death and oxidative stress. G protein-coupled receptor kinase-2 (GRK2) is a key molecule involved in myocardial I/R injury. In vivo and in vitro experiments showed that LBP inhibited the upregulation of GRK2 expression induced by I/R injury, which was related to the antiapoptotic effect of LBP. In addition, we found that LBP partially restored I/R-induced mitochondrial fission/fusion imbalance, as well as levels of phosphorylated protein kinase B (p-AKT) and phosphorylated endothelial cell nitric oxide synthase (p-eNOS), and this restorative effect could be attenuated by overexpression of GRK2. Overall, our findings suggest that LBP antagonizes cardiomyocyte apoptosis by inhibiting the upregulation of GRK2 induced by I/R injury and saves mitochondrial fission/fusion imbalance and AKT/eNOS signaling. This study may provide new ideas for the study of I/R injury and the rational application of the herbal medicine LBP.

Aerobic Exercise Restores Aging-Associated Reductions in Arterial Adropin Levels and Improves Adropin-Induced Nitric Oxide-Dependent Vasorelaxation.

BackgroundAdropin is a peptide hormone that promotes nitric oxide (NO) production via activation of endothelial NO synthase (eNOS) in endothelial cells. Its circulating levels are reduced with aging and increased with aerobic exercise training (AT). Using a mouse model, we hypothesized that AT restores aging‐associated reductions in arterial and circulating adropin and improves adropin‐induced NO‐dependent vasorelaxation. Further, we hypothesized these findings would be consistent with data obtained in elderly humans.Methods and ResultsIn the animal study, 50‐week‐old SAMP1 male mice that underwent 12 weeks of voluntary wheel running, or kept sedentary, were studied. A separate cohort of 25‐week‐old SAMP1 male mice were used as a mature adult sedentary group. In the human study, 14 healthy elderly subjects completed an 8‐week AT program consisting of 45 minutes of cycling 3 days/week. In mice, we show that advanced age is associated with a decline in arterial and circulating levels of adropin along with deterioration of endothelial function, arterial NO production, and adropin‐induced vasodilation. All these defects were restored by AT. Moreover, AT‐induced increases in arterial adropin were correlated with increases in arterial eNOS phosphorylation and NO production. Consistently with these findings in mice, AT in elderly subjects enhanced circulating adropin levels and these effects were correlated with increases in circulating nitrite/nitrate (NOx) and endothelial function.ConclusionsChanges in arterial adropin that occur with age or AT relate to alterations in endothelial function and NO production, supporting the notion that adropin should be considered a therapeutic target for vascular aging.RegistrationURL: https://www.umin.ac.jp; Unique identifier: UMIN000035520.

Non‐Nutritive Sweeteners Reduce Endothelial Cell Nitric Oxide Synthase Activity and Nitric Oxide Production

Non-nutritive sweeteners (NNS), such as sucralose and acesulfame potassium (ace K), offer low-calorie alternatives to traditional sweeteners, such as glucose and fructose. Substituting NNSs for these traditional sweeteners, particularly in popular beverages such as soft drinks, has been shown to help mitigate some, not all, of sugar-associated cardiometabolic risk. However, the effect of NNSs on endothelial cell function are not fully understood. The experimental aim of this study was to determine if sucralose and ace k affects endothelial cell inflammation, apoptosis and nitric oxide (NO) production, key functional properties of vascular health. Human umbilical vein endothelial cells were cultured and treated with concentrations of sucralose (0.169mg/mL) and/or ace k (0.127mg/mL) that are equivalent to the amount in one 12 oz can of diet soda (i.e. Diet Coke with Splenda or Pepsi One) for 24 h. Neither sucralose, ace k, nor sucralose + ace k significantly altered markers of endothelial cell inflammation. Total cellular expression of the primary inflammation transcription factor, nuclear factor kappa B (NF-kB) was not different in endothelial cells treated with sucralose (46.0±10.6 AU), ace k (41.3±17.4 AU), sucralose + ace k (47.9±15.4 AU) or control (50.8±15.7 AU). Activated NF-kB was also similar in cells treated with sucralose (130.1±29.6 AU), ace k (122.0±32.4 AU), sucralose + ace k (118.4±33.5 AU) and control (116.7±16.3 AU). In addition, NNSs did not affect endothelial cell apoptosis. Total caspase 3 expression, a key apoptosis protein, was not significantly different between sucralose (65.1±14.0 AU), ace k (78.8±5.4 AU), sucralose + ace k (59.0±9.4 AU) and control (88.1±14.7 AU) conditions. Of note, the expression of active caspase 3 was also not significantly different between sucralose (13.0±3.0 AU), ace k (12.2±3.2 AU), sucralose + ace k (11.8±3.3 AU) and control (11.7±1.6 AU). Endothelial nitric oxide synthase (eNOS) is fundamental to endothelial cell function and the production of NO. Total eNOS expression was not significantly different in cells treated with sucralose (155.5±22.6 AU), ace k (260.9±53.5 AU), sucralose + ace k (175.3±36.3 AU) or control (245.3±27.0 AU); however, sucralose (180.9±36.9 AU), ace k (208.0±40.2 AU) and sucralose + ace k (115.1±17.5 AU) markedly lowered (P<0.05) eNOS activation (phosphorylated-(p)eNOS serine 1177) compared with the control condition (375.9±74.6 AU). Concordantly, NO production was significantly reduced in cells treated with sucralose (7.5±0.4 mmol/L), ace k (7.9±0.2 mmol/L), and sucralose + ace k (6.9±0.2 mmol/L) compared with control (9.1±0.5 mmol/L). The effect of NSSs on p-eNOS and NO production was similar across the conditions. In conclusion, NNSs commonly contained in artificially sweetened beverages do not affect specific endothelial cell inflammation or apoptotic proteins, however NNSs adversely affect endothelial cell nitric oxide production. Future studies are needed to determine whether NNS are associated with impaired endothelial vasodilator dysfunction and the potential vascular consquences of reduced NO bioavailabilty.

How does ascorbate improve endothelial dysfunction? - A computational analysis

Low levels of ascorbate (Asc) are observed in cardiovascular and neurovascular diseases. Asc has therapeutic potential for the treatment of endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability and increased oxidative stress in the vasculature. However, the potential mechanisms remain poorly understood for the Asc mitigation of endothelial dysfunction. In this study, we developed an endothelial cell based computational model integrating endothelial cell nitric oxide synthase (eNOS) biochemical pathway with downstream reactions and interactions of oxidative stress, tetrahydrobiopterin (BH4) synthesis and biopterin ratio ([BH4]/[TBP]), Asc and glutathione (GSH). We quantitatively analyzed three Asc mediated mechanisms that are reported to improve/maintain endothelial cell function. The mechanisms include the reduction of •BH3 to BH4, direct scavenging of superoxide (O2•−) and peroxynitrite (ONOO−) and increasing eNOS activity. The model predicted that Asc at 0.1–100 μM concentrations improved endothelial cell NO production, total biopterin and biopterin ratio in a dose dependent manner and the extent of cellular oxidative stress. Asc increased BH4 availability and restored eNOS coupling under oxidative stress conditions. Asc at concentrations of 1–10 mM reduced O2•− and ONOO− levels and could act as an antioxidant. We predicted that glutathione peroxidase and peroxiredoxin in combination with GSH and Asc can restore eNOS coupling and NO production under oxidative stress conditions. Asc supplementation may be used as an effective therapeutic strategy when BH4 levels are depleted. This study provides detailed understanding of the mechanism responsible and the optimal cellular Asc levels for improvement in endothelial dysfunction.

β-Arrestin2 is a critical component of the GPCR–eNOS signalosome

Endothelial cell nitric oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in endothelial cells, is regulated by complex posttranslational mechanisms. Sinusoidal portal hypertension, a disorder characterized by liver sinusoidal endothelial cell (SEC) injury with resultant reduced eNOS activity and NO production within the liver, has been associated with defects in eNOS protein-protein interactions and posttranslational modifications. We and others have previously identified novel eNOS interactors, including G protein-coupled receptor (GPCR) kinase interactor 1 (GIT1), which we found to play an unexpected stimulatory role in GPCR-mediated eNOS signaling. Here we report that β-arrestin 2 (β-Arr2), a canonical GPCR signaling partner, localizes in SECs with eNOS in a GIT1/eNOS/NO signaling module. Most importantly, we show that β-Arr2 stimulates eNOS activity, and that β-Arr2 expression is reduced and formation of the GIT1/eNOS/NO signaling module is interrupted during liver injury. In β-Arr2-deficient mice, bile duct ligation injury (BDL) led to significantly reduced eNOS activity and to a dramatic increase in portal hypertension compared to BDL in wild-type mice. Overexpression of β-Arr2 in injured or β-Arr2-deficient SECs rescued eNOS function by increasing eNOS complex formation and NO production. We also found that β-Arr2-mediated GIT1/eNOS complex formation is dependent on Erk1/2 and Src, two kinases known to interact with and be activated by β-Arr2 in response to GCPR activation. Our data emphasize that β-Arr2 is an integral component of the GIT1/eNOS/NO signaling pathway and have implications for the pathogenesis of sinusoidal portal hypertension.

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

ObjectivePulmonary 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.MethodsDoppler 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 (AT1R), AT2R, 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.ResultsPSGL‐1−/− mice showed lung vessel wall remodeling and a reduced mean ± SD expression of pulmonary AT2R (expression ratio [relative to β‐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.ConclusionThe 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.

Stachydrine Mediates Rapid Vascular Relaxation: Activation of Endothelial Nitric Oxide Synthase Involving AMP-Activated Protein Kinase and Akt Phosphorylation in Vascular Endothelial Cells.

Stachydrine (STA) is a constituent of citrus fruits and Leonurus heterophyllus Sweet. In the present study, we established that STA caused acute endothelium-dependent relaxation. The vascular action of STA was mediated by nitric oxide (NO) via cyclic guanosine monophosphate. Mechanistically, STA activated AMP-activated protein kinase (AMPK), protein kinase B/Akt, and endothelial NO synthase (eNOS) in vascular endothelial cells (ECs). AMPK inhibition by compound C blocked STA-induced Akt/eNOS phosphorylation, suggesting that AMPK is the upstream of Akt and eNOS. Inhibition of Akt by MK2206 blocked STA-stimulated eNOS phosphorylation without altering AMPK phosphorylation. Furthermore, we showed that STA activated AMPK via the induction of liver kinase B1 phosphorylation. These results indicated that STA can induce eNOS phosphorylation and vasorelaxation by regulating the interplay between AMPK and Akt pathways in ECs. These findings further highlighted the potential of STA as a nutritional factor in the beneficial effects of fruit intake in preventing the endothelial dysfunction-related metabolic cardiovascular diseases.

Inhibition of acid sphingomyelinase activity ameliorates endothelial dysfunction in db/db mice.

Acid sphingomyelinase (aSMase) plays an important role in endothelial dysfunction. Here, we show that elevated aSMase activity and ceramide content were reduced by desipramine treatment in diabetic animals. The inhibitor of aSMase, desipramine, improved vascular dysfunction in db/db mice. High glucose (HG)-induced up-regulation of aSMase activity and ceramide levels were restored by treatment with aSMase siRNA or desipramine in endothelial cells. In addition, aSMase siRNA or desipramine treatment increased the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in diabetic mouse aortas and aortic endothelial cells with HG.These results indicate that inhibition of aSMase/ceramide pathway improves endothelium-dependent vascular relaxation (EDR) largely through regulating the eNOS/NO pathway in diabetic animals.

Clinical value of anti-oxidative stress biomarkers for diagnosing in-stent restenosis and in-stent reocclusion after coronary stent implantation in advanced-aged patients

Objective To study the clinical value of anti-oxidative stress biomarkers for diagnosing in-stent restenosis and in-reocclusion after coronary stent implantation in aged patients. Methods A total of 72 advanced-aged patients with in-stent restenosis and in-stent reocclusion after coronary stent implantation were successively recruited in this retrospective study from February 2010 to November 2017.Changes in serum superoxide dismutase 3(SOD3), nitric oxide(NO), endothelial cell nitric oxide synthase(eNOS)and malondialdehyde(MDA)levels were measured. Results Serum 1evels of SOD3, NO and eNOS decreased and serum MDA levels were elevated in advanced-aged patients with in-stent restenosis.There were significant differences in serum levels of SOD3, NO, eNOS and MDA between the advanced-aged patients without in-stent restenosis and the advanced-aged patients with multivessel in-stent restenosis or reocclusion[(20.0±3.2)×103U/L vs.(10.9±3.9)×103U/L, (61.2±14.2)μmol/L vs.(28.3±17.2)μmol/L, (75.9±24.7)ng/L vs.(33.0±19.6)ng/L, (2.2±1.4)nmol/L vs.(11.7±3.1)nmol/L, respectively, P<0.01]. Patients with 50-69% restenosis had higher serum levels of SOD3, NO and eNOS and lower levels of MDA than patients with 100% restenosis[(21.3±2.9)×103U/L vs.(10.3±4.0)×103U/L, (59.7±16.7)μmol/L vs.(38.3±16.3)μmol/L, (74.5±21.1)ng/L vs.(41.9±26.8)ng/L, (2.6±3.9 nmol/L)vs.(10.1±3.1)nmol/L, respectively, P<0.01]. Patients with left ventricular ejection fraction(LVEF)≥55% had higher serum levels of SOD3, NO and eNOS and lower levels of MDA than patients with LVEF<30%[(21.0±4.1)×103U/L vs.(5.3±1.9)×103U/L, (60.1±14.2)μmol/L vs.(29.0±13.2)μmol/L, (74.7±25.1)ng/L vs.(39.3±20.3)ng/L, (2.3±1.5)nmol/L vs.(10.0±3.9)nmol/L, respectively, P<0.01]. Serum levels of SOD3, NO and eNOS were higher and MDA levels were lower in patients with New York Heart Association(NYHA)Class I than in patients with NYHA Class IV[(22.1±3.5)×103U/L vs.(9.7±2.9)×103U/L, (62.9±13.9)μmol/L vs.(24.9±13.3)μmol/L, (76.7±26.7)ng/L vs.(41.9±21.5)ng/L, (2.7±1.9)nmol/L vs.(8.7±3.8)nmol/L, respectively, P<0.01]. Conclusions Serum level changes of anti-oxidative stress biomarkers such as SOD3, NO and eNOS may have clinical value in diagnosing in-stent restenosis and in-reocclusion after coronary stent implantation in aged patients. Key words: Coronary restenosis; Superoxide dismutase; Nitric oxide Synthase

Platelet-rich plasma enhances adipose-derived stem cell-mediated angiogenesis in a mouse ischemic hindlimb model.

AIMTo evaluate the angiogenic effect of platelet-rich plasma (PRP)-preconditioned adipose-derived stem cells (ADSCs) both in vitro and in a mouse ischemic hindlimb model.METHODSADSCs were divided based on culture medium: 2.5% PRP, 5% PRP, 7.5% PRP, and 10% PRP. Cell proliferation rate was analyzed using the MTS assay. The gene expression of CD31, vascular endothelial growth factor, hypoxia-inducible factors, and endothelial cell nitric oxide synthase was analyzed using reverse transcription polymerase chain reaction. Cell markers and structural changes were assessed through immunofluorescence staining and the tube formation assay. Subsequently, we studied the in vivo angiogenic capabilities of ADSCs by a mouse ischemic hindlimb model.RESULTSThe proliferation rate of ADSCs was higher in the 2.5%, 5%, and 7.5% PRP groups. The expression of hypoxia-inducible factor, CD31, vascular endothelial growth factor, and endothelial cell nitric oxide synthase in the 5% and 7.5% PRP groups increased. The 5%, 7.5%, and 10% PRP groups showed higher abilities to promote both CD31 and vascular endothelial growth factor production and tubular structure formation in ADSCs. According to laser Doppler perfusion scan, the perfusion ratios of ischemic limb to normal limb were significantly higher in 5% PRP, 7.5% PRP, and human umbilical vein endothelial cells groups compared with the negative control and fetal bovine serum (FBS) groups (0.88 ± 0.08, 0.85 ± 0.07 and 0.81 ± 0.06 for 5%, 7.5% PRP and human umbilical vein endothelial cells compared with 0.42 ± 0.17 and 0.54 ± 0.14 for the negative control and FBS, P < 0.01).CONCLUSIONPRP-preconditioned ADSCs presented endothelial cell characteristics in vitro and significantly improved neovascularization in ischemic hindlimbs. The optimal angiogenic effect occurred in 5% PRP- and 7.5% PRP-preconditioned ADSCs.

Elevated arterial shear rate increases indexes of endothelial cell autophagy and nitric oxide synthase activation in humans.

Continuous laminar shear stress increases the process of autophagy, activates endothelial nitric oxide (NO) synthase phosphorylation at serine 1177 (p-eNOSS1177), and generates NO in bovine and human arterial endothelial cells (ECs) compared with static controls. However, the translational relevance of these findings has not been explored. In the current study, primary ECs were collected from the radial artery of 7 men using sterile J-wires before (Pre) and after (Post) 60 min of rhythmic handgrip exercise (HG) performed with the same arm. After ECs were identified by positive costaining for vascular endothelial cadherin and 4',6'-diamidino-2-phenylindole, immunofluorescent antibodies were used to assess indices of autophagy, NO generation, and superoxide anion (O2·-) production. Commercially available primary human arterial ECs were stained and processed in parallel to serve as controls. All end points were evaluated using 75 ECs from each subject. Relative to Pre-HG, HG elevated arterial shear rate ( P < 0.05) ~3-fold, whereas heart rate, arterial pressure, and cardiac output were not altered. Compared with values obtained from ECs Pre-HG, Post-HG ECs displayed increased ( P < 0.05) expression of p-eNOSS1177, NO generation, O2·- production, BECLIN1, microtubule-associated proteins 1A/1B light chain 3B, autophagy-related gene 3, and lysosomal-associated membrane protein 2A and decreased ( P < 0.05) expression (i.e., enhanced degradation) of the adaptor protein p62/sequestosome-1. These novel findings provide evidence that elevated arterial shear rate associated with functional hyperemia initiates autophagy, activates p-eNOSS1177, and increases NO and O2·- generation in primary human ECs. NEW & NOTEWORTHY Previously, our group reported in bovine arterial and human arterial endothelial cells (ECs) that shear stress initiates trafficking of the autophagosome to the lysosome and increases endothelial nitric oxide (NO) synthase phosphorylation at serine 1177, NO generation, and O2·- production. Here, the translational relevance of these findings is documented. Specifically, functional hyperemia induced by rhythmic handgrip exercise elevates arterial shear rate to an extent that increases indices of autophagy, NO generation, and O2·- production in primary arterial ECs collected from healthy men.

Protective Effect of Dexamethasone on Lipopolysaccharide-Induced Inhibition of Contractile Function of Isolated Lymphatic Vessels and Nodes.

LPS has an inhibitory effect on contractile activity of bovine mesenteric lymphatic vessels and nodes and causes a pronounced decrease in the tone and phase contractions. The selective inhibitor of inducible NO synthase, 1400W, and cyclooxygenase-2 selective inhibitor, dynastat, significantly attenuated the inhibitory effect of LPS. Dexamethasone interferes with the inhibitory effect of LPS on bovine lymphatic vessels and nodes. It was concluded that LPS stimulates expression of inducible NO synthase and cyclooxygenase-2 in endothelial and smooth muscle cells of lymphatic vessels and nodes. Dexamethasone has a pronounced protective effect on the contractile function of lymphatic vessels and nodes affected by LPS and suppresses the expression of inducible NO synthase and cyclooxygenase-2.

Effect of SFRP5 (Secreted Frizzled–Related Protein 5) on the WNT5A (Wingless-Type Family Member 5A)-Induced Endothelial Dysfunction and Its Relevance With Arterial Stiffness in Human Subjects

SFRP5 (secreted frizzled-related protein 5) is an endogenous inhibitor of WNT5A (wingless-type family member 5a), which has been implicated in atherosclerosis. However, contradictory results have been reported about the role of SFRP5 in atherosclerosis. We aimed to investigate whether SFRP5 could restore WNT5A-induced endothelial dysfunction in vitro and ex vivo. In addition, we sought to determine whether the serum concentration of SFRP5 is associated with atherosclerosis in humans. We measured endothelium-dependent vasorelaxation in the isolated thoracic aorta of Sprague-Dawley rats. In addition, we measured intracellular nitric oxide (NO) in human endothelial cells. The protein abundance of total and phosphorylated JNK (c-Jun N-terminal kinase), AKT (protein kinase B), and endothelial NO synthase was analyzed in human endothelial cells. Circulating SFRP5 and WNT5A levels and brachial-ankle pulse wave velocity were measured in 282 human subjects with type 2 diabetes mellitus. SFRP5 dose dependently restored Wnt5-induced impaired vasorelaxation in rat thoracic aorta by an endothelial NO synthase-dependent mechanism. SFRP5 treatment restored the WNT5A-induced reduction of NO production via endothelial NO synthase in human endothelial cells. WNT5A-induced changes in the phosphorylation of JNK, AKT, and endothelial NO synthase were ameliorated with SFRP5 administration. In humans with type 2 diabetes mellitus, the serum SFRP5 concentration positively correlated with brachial-ankle pulse wave velocity (r=0.146; P=0.024). Multivariate linear regression analysis demonstrated that the serum SFRP5 concentration was independently associated with brachial-ankle pulse wave velocity after adjustment for potential confounders [B (SE)=7.40 (3.35); P=0.028]. Our data suggest the possible compensatory action of SFRP5 against atherosclerosis under conditions of metabolic dysfunction.

Rhythmic handgrip exercise elevates arterial shear‐rate and increases indices of endothelial cell autophagy and nitric oxide synthase activation in humans

Autophagy is a process whereby damaged cellular cargo is delivered to the lysosome for degradation and eventual recycling or removal. Knowledge concerning the importance of autophagy to endothelial cell (EC) metabolism is evolving. Previously we reported that 180‐min × 20 dyne · cm 2 shear‐stress initiates autophagy, activates EC nitric oxide (NO) synthase (eNOS) at serine 1177 (p‐eNOSS1177), and generates NO in immortalized bovine and human arterial endothelial cells (ECs) (Bharath et al., Arterioscler Thromb Vasc Biol, 2017). Importantly, when EC autophagy was repressed using genetic or pharmacological procedures, the ability of shear‐stress to generate NO was prevented. Here we report data from the first step to eventually determine whether these findings obtained in vitro can be translated to primary human ECs. After familiarization with laboratory procedures (visit 1), five male subjects (23±3 yr) completed a maximal voluntary contraction (MVC) test and a flow‐mediated vasodilation (FMD) assessment during a second visit (visit 2). Within 1‐week, a 20 g catheter was placed into the radial artery (RA) during visit 3. Next, ECs were collected via j‐wire biopsy from the RA catheter, transferred to dissociation buffer, recovered by washing and centrifugation, placed onto previously prepared glass slides, and frozen at −80°C. Thirty‐min after collecting pre‐exercise ECs, subjects performed rhythmic handgrip exercise for 60‐min at a contraction intensity that elevated (p&lt;0.05) arterial shear‐rate 2.7 ± 0.3‐fold from baseline. Shear‐rate [8Vmean/brachial artery (BA) diameter] was obtained by direct and continuous assessment of BA diameter, BA blood flow velocity (Vmean), and BA blood flow [(Vmeanπ (vessel diameter/2)2 × 60)] using Doppler ultrasound. Heart rate (HR, beats/min; 3‐lead ECG), stroke volume (SV, ml/beat), cardiac output (CO, L/min), and mean arterial pressure (MAP, mmHg; automated plethysmography), respectively, were not different when pre‐exercise values (56±1, 101±6, 5.6±0.3, and 91±3) were compared to 60‐min values (60±2, 104±6, 6.2±0.4, and 94±2). At 60‐min of handgrip exercise, ECs were collected from the RA, prepared, and stored as described. Using quantitative immunofluorescence, primary ECs (75 ECs per endpoint) were identified by positive co‐staining for VE‐Cadherin and DAPI via confocal microscopy. Immortalized human arterial endothelial cells (P4–6) were processed and stained in parallel using identical procedures to serve as fluorescence intensity controls. Relative to pre‐exercise values, ECs obtained at 60‐min displayed increased expression of beclin‐1 (p&lt;0.01), microtubule associated protein light chain 3 (LC3 II; p=0.05), autophagy‐related gene 3 (Atg3; p=0.06), lysosomal associated membrane protein 2a (LAMP2a; p&lt;0.004), and p‐eNOSS1177 (p&lt;0.02), and decreased expression (i.e., enhanced degradation) of the adapter protein p62/SQSTM1 (p&lt;0.02). Taken together, these findings provide novel evidence in primary human ECs that elevated shear‐rate evoked by functional hyperemia initiates autophagy and activates p‐eNOSS1177. Ongoing studies in our laboratory are determining whether this response is dysregulated in older humans and in patients with type 2 diabetes, conditions associated with arterial dysfunction.Support or Funding InformationAmerican Heart Association (AHA)17POST33670663 to SKP; AHA 16GRNT31050004 and National Institutes of Health NIH:RO3AGO52848 to JDS; Veteran Affairs Rehabilitation Research and Development Grant IK2RX001215 CDA2 and American Heart Association Grant 14SDG1850039 to JDT.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SNPs, linkage disequilibrium, and chronic mountain sickness in Tibetan Chinese.

Chronic mountain sickness (CMS) is estimated at 1.2% in Tibetans living at the Qinghai–Tibetan Plateau. Eighteen single-nucleotide polymorphisms (SNPs) from nine nuclear genes that have an association with CMS in Tibetans have been analyzed by using pairwise linkage disequilibrium (LD). The SNPs included are the angiotensin-converting enzyme (rs4340), the angiotensinogen (rs699), and the angiotensin II type 1 receptor (AGTR1) (rs5186) from the renin–angiotensin system. A low-density lipoprotein apolipoprotein B (rs693) SNP was also included. From the hypoxia-inducible factor oxygen signaling pathway, the endothetal Per-Arnt-Sim domain protein 1 (EPAS1) and the egl nine homolog 1 (ENGL1) (rs480902) SNPs were included in the study. SNPs from the vascular endothelial growth factor (VEGF) signaling pathway included are the v-akt murine thymoma viral oncogene homolog 3 (rs4590656 and rs2291409), the endothelial cell nitric oxide synthase 3 (rs1007311 and rs1799983), and the (VEGFA) (rs699947, rs34357231, rs79469752, rs13207351, rs28357093, rs1570360, rs2010963, and rs3025039). An increase in LD occurred in 40 pairwise comparisons, whereas a decrease in LD was found in 55 pairwise comparisons between the controls and CMS patients. These changes were found to occur within and between signaling pathways, which suggests that there is an interaction between SNP alleles from different areas of the genome that affect CMS.

Low Shear Stress Inhibited Endothelial Cell Autophagy Through TET2 Downregulation.

Low shear stress plays a crucial role in the initiation and progression of atherosclerotic lesions. However, the detailed mechanisms of these processes remain unclear. In this study, the effect of low shear stress on endothelial cell autophagy and its potential mechanism were investigated. Results showed autophagy dysfunction and ten-eleven translocation 2 (TET2) protein downregulation during atherosclerotic lesion progression. Autophagic markers BECLIN 1 and LC3II/LC3I under low shear stress (5dyne/cm(2)) obviously decreased compared with those under physiological shear stress (15dyne/cm(2)), whereas autophagic substrate p62 increased. TET2 expression was also downregulated under low shear stress. Endothelial cell autophagy was improved with TET2 overexpression but was impaired by TET2 siRNA treatment. Moreover, TET2 overexpression upregulated the expression of endothelial cell nitric oxide synthase (eNOS) and downregulated the expression of endothelin-1 (ET-1). TET2siRNA further attenuated eNOS expression and stimulated ET-1 expression. Overall, the results showed that low shear stress downregulated endothelial cell autophagy by impaired TET2 expression, which might contribute to theatherogenic process.

Long-Term Gestational Hypoxia Modulates Expression of Key Genes Governing Mitochondrial Function in the Perirenal Adipose of the Late Gestation Sheep Fetus.

We previously reported that long-term hypoxia (LTH) increases expression of brown adipose tissue (BAT) genes in the perirenal adipose in the ovine fetus. The mechanisms with which hypoxia mediates the enhanced BAT phenotype are unresolved. This study was designed to examine the effects of LTH on (1) the expression of endothelial cell nitric oxide synthase (eNOS) and (2) indicators of mitochondrial biogenesis (transcription factors mitochondrial transcription factor A (mtTFA), nuclear respiratory factor (NRF) 1, and NRF-2; cytochrome c oxidase (COX) I, II, and IV and mitochondrial DNA content). Pregnant ewes were maintained at high altitude (3820 m) from ∼40 to 137 to 140 days of gestation and perirenal adipose was collected from normoxic control and LTH fetuses. There was no effect of LTH on fetal body weight or perirenal adipose mass. Long-term hypoxia increased (P < .05) perirenal eNOS and phospho-eNOS, messenger RNA (mRNA) for NRF1, NRF-2, mtTFA as well as COX-I, COX-II, and COX-IV mRNA. In contrast, mRNA for 2 markers for cellular proliferation (Ki67 and proliferating cell nuclear antigen [PCNA]) was lower in perirenal adipose from LTH fetuses compared to controls (P < .05), while mitochondrial to nuclear DNA ratio did not differ between groups. In conclusion, nitric oxide may function as a mechanism via which LTH enhances the BAT phenotype in fetal sheep prior to birth. Although there is an apparent increase in genes supporting mitochondrial function and adaptive thermogenesis in response to LTH, there does not appear to be an increased mitochondrial biogenesis per se. Such adaptive changes may provide a mechanism for the prominence of the BAT phenotype observed in the late gestation LTH fetus.