Metformin promotes angiogenesis and functional recovery in aged mice after spinal cord injury by adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

Treatment with metformin can lead to the recovery of pleiotropic biological activities after spinal cord injury.However,its effect on spinal cord injury in aged mice remains unclear.Considering the essential role of angiogenesis during the regeneration process,we hypothesized that metformin activates the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway in endothelial cells,thereby promoting microvascular regeneration in aged mice after spinal cord injury.In this study,we established young and aged mouse models of contusive spinal cord injury using a modified Allen method.We found that aging hindered the recovery of neurological function and the formation of blood vessels in the spinal cord.Treatment with metformin promoted spinal cord microvascular endothelial cell migration and blood vessel formation in vitro.Furthermore,intraperitoneal injection of metformin in an in vivo model promoted endothelial cell proliferation and increased the density of new blood vessels in the spinal cord,thereby improving neurological function.The role of metformin was reversed by compound C,an adenosine monophosphate-activated protein kinase inhibitor,both in vivo and in vitro,suggesting that the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway likely regulates metformin-mediated angiogenesis after spinal cord injury.These findings suggest that metformin promotes vascular regeneration in the injured spinal cord by activating the adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway,thereby improving the neurological function of aged mice after spinal cord injury.

Endothelial nitric oxide synthase deficiency influences normal cell cycle progression and apoptosis in trabecular meshwork cells

AIM： To clarify how the endothelial nitric oxide synthase （eNOS, NOS3） make effect on outflow facility through the trabecular meshwork （TM）. METHODS： Inhibition of NOS3 gene expression in human TM cells were conducted by three siRNAs. Then the mRNA and protein levels of NOS3 in siRNA-treated and negative control （NC） cells were determined, still were the collagen, type IV, alpha 1 （COL4A1） and fibronectin 1 by real-time PCR and Western blot analysis. In addition, NOS3 concentrations in culture supernatant fluids of TM cells were measured. Cell cycle and cell apoptosis analysis were performed using flow cytometry. RESULTS： The mRNA level of NOS3 was decreased by three different siRNA interference, similar results were obtained not only of the relative levels of NOS3 protein, but also the expression levels of COL4A1 and fibronectin 1. The number of cells in S phase was decreased, while contrary result was obtained in G2 phase. The number of apoptotic cells in siRNA-treated groups were significant increased compared to the NC samples. CONCLUSION： Abnormal NOS3 expression can make effects on the proteins levels of extracellular matrix component （e.g. fibronectin 1 and COL4A1）. Reduced NOS3 restrains the TM cell cycle progression at the G2/ M-phase transition and induced cell apoptosis.

Expression of Endothelial Nitric Oxide Synthase Traffic Inducer in the Placenta of Pregnancy Induced Hypertension

The expression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） in the placenta of the patients with pregnancy induced hypertension （PIH） was detected and its role in the pathogenesis of PIH was studied. The pathological changes in placental vessels were observed by HE staining. NO2-/NO3- , the stable metabolic end products of NO, was measured with nitrate reductase. The eNOS activity in placental tissues was assayed by spectrophotometry. Western blot analysis was applied to detect NOSTRIN expression. The incidence of thickening and fibronoid necrosis of placental vessels was significantly higher in women with PIH than in the normal group （P〈 0.01）. The levels of placental NO2-/NO3- in PIH patients （27. 53±7.48 μmol/mg） were significantly lower than in normal group （54. 27±9.53 μmol/mg, P〈0.01）. The activity of eNOS was significantly decreased in PIH group （12. 826±3.61 U/mg） as compared with that in normal group （21. 72±3.83 U/mg, P〈0.01）. Western blot analysis revealed that both groups expressed 58 kD NOSTRIN, but the protein level was significantly higher in women with PIH than in the normal group （P〈0.01）. A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in placental tissue of women with PIH （r=-0.57, P〈0.01）. It was concluded that the level of NOSTRIN expression in placenta of women with PIH was increased, which may play an important role in the pathogenesis of PIH.

The emerging role of nitric oxide in the synaptic dysfunction of vascular dementia

With an increase in global aging,the number of people affected by cerebrovascular diseases is also increasing,and the incidence of vascular dementia-closely related to cerebrovascular risk-is increasing at an epidemic rate.However,few therapeutic options exist that can markedly improve the cognitive impairment and prognosis of vascular dementia patients.Similarly in Alzheimer’s disease and other neurological disorders,synaptic dysfunction is recognized as the main reason for cognitive decline.Nitric oxide is one of the ubiquitous gaseous cellular messengers involved in multiple physiological and pathological processes of the central nervous system.Recently,nitric oxide has been implicated in regulating synaptic plasticity and plays an important role in the pathogenesis of vascular dementia.This review introduces in detail the emerging role of nitric oxide in physiological and pathological states of vascular dementia and summarizes the diverse effects of nitric oxide on different aspects of synaptic dysfunction,neuroinflammation,oxidative stress,and blood-brain barrier dysfunction that underlie the progress of vascular dementia.Additionally,we propose that targeting the nitric oxide-sGC-cGMP pathway using certain specific approaches may provide a novel therapeutic strategy for vascular dementia.

Association study of the endothelial nitric oxide synthase gene polymorphisms with essential hypertension in northern Han Chinese

Background Nitric oxide （NO） synthesized by endothelial nitric oxide synthase （eNOS） plays an important role in both the regulation of endothelial function and the control of blood pressure. Up to now, there has been conflicting data regarding the association between three clinically relevant polymorphisms （T-786C, intron4b/a and G894T） of the eNOS gene and essential hypertension.

Association of endothelial nitric oxide synthase polymorphisms with an increased risk of erectile dysfunction

The purpose of our meta-analysis is to examine the associations between three single nucieotide polymorphisms of endothelial nitric oxide synthase （eNOS） gene, G894T, intron 4 and T-786C, and the risk of erectile dysfunction. An electronic database search was performed to identify case-control studies reporting the association between single nucleotide polymorphisms of eNOS gene and erectile dysfunction. Stringent inclusion and exclusion criteria were employed to select high-quality studies for this meta-analysis. Comprehensive Meta-analysis 2.0 software （Biostat Inc., Englewood, New Jersey, USA） was used for statistical analysis of the data extracted from the selected studies. From the initial 203 articles retrieved from database search, this meta-analysis finally selected 12 high-quality case-control studies that conformed to our inclusion criteria. The 12 studies contained a total of 1962 patients with erectile dysfunction and 1752 healthy controls. The results of our meta-analysis showed that G894T correlated with an increased risk erectile dysfunction under both the allele and dominant models （allele： OR = 1.556, 95% CI -- 1.064-2.275, P = 0.023; dominant： OR = 1.613, 95% CI = 1.050-2.476, P = 0.029）. A similar association was found between T-786C and erectile dysfunction under the allele model （OR = 1.679, 95% CI = 1.341-2.102, P 〈 0.001）, but not under the dominant model （all P 〉 0.05）. Our meta-analysis showed that the two single nucleotide polymorphisms in eNOS gene, G894T and T-786C, are strongly associated with the risk of erectile dysfunction.

Calpa,n inhibition improves erectile function in diabetic mice via upregulating endothelial nitric oxide synthase expression and reducing apoptosis

Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction （ED） in mice, Thirty-eight-week-old male C57BIJ6J mice were divided into three groups： （1） nondiabetic control group, （2） diabetic mice ＋ vehicle group, and （3） diabetic mice ＋ MDL28170 （an inhibitor of calpain） group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg-1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase （eNOS）-nitric oxide （NO）-cyclic guanosine monophosphate （cGMP） pathway. Terminal deoxynucleotidyl transferase 2＇-deoxyuridine 5＇-triphosphate nick end labeling （TUNEL） staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, caipain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.

INSULIN INDUCES NITRIC OXIDE PRODUCTION IN BOVINE AORTIC ENDOTHELIAL CELLS

ffeStun6. ObjeCtif Dens cette dtude nons avons ewilue l' effet de l' insuline sur ba Proliferation cellulaire, liberationd' oxide nitrique et l' expression gdrktique de synthase d' oxide nitrique dens la cellule endotheliale aortique bovine. methIn mitogdthe est evalude per la ndthae M7T. In Production de NO dans ie alga en culture est determine per la reactionGness. In technique quantitative RT/PCR est utility pour quantifier ie niveau de sWthase d' oxide nitrique mRNA dens la cellule endotheliale aortique...

New progress in understanding roles of nitric oxide during hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury(HIRI)is a major clinical cause of morbidity and mortality in liver surgery and transplantation.Many studies have found that nitric oxide(NO)plays an important role in the HIRI and its increase or decrease can affect the progression and outcome of HIRI.However,the role of NO in HIRI is controversial and complicated.NO derived by endothelial NO synthase(eNOS)shows a protective role in HIRI,while excessive NO derived by inducible NO synthase(iNOS)accelerates inflammation and increases oxidative stress,further aggravating HIRI.Nevertheless,the overexpression of eNOS may exacerbate HIRI and iNOS-derived NO in some cases reduces HIRI.Here we review the new progress in the understanding of the roles of NO during HIRI:(1)NO possesses different roles in HIRI by increasing NO bioavailability,down-regulating leukotriene C4 synthase,inhibiting the activation of the nuclear factorκB(NFκB)pathway,enhancing cell autophagy,and reducing inflammatory cytokines and reactive oxygen species(ROS).And NO has both protective and deleterious effects by regulating apoptotic factors;(2)eNOS promotes NO production and suppresses its own overexpression,exerting a hepatoprotective effect reversely.Its activation is regulated by the PI3K/Akt and KLF2/AMPK pathways;and(3)iNOS derived NO mainly has deteriorating effects on HIRI,while it may have a protective function under some conditions.Their expression should reach a balance to reduce the adverse side and make NO protective in the treatment of HIRI.Thus,it can be inferred that NO modulating drugs may be a new direction in the treatment of HIRI or may be used as an adjunct to mitigate HIRI for the purpose of protecting the liver.

Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide

Background The vascular endothelial growth factor （VEGF） is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 （ICAM-1） is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells （ECs） is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs. Methods Bovine retinal ECs （BRECs） were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF （100 ng/ml） and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase （eNOS） were detected using Western blotting. Griess reaction was used to detect nitric oxide （NO）. Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C （PKC） altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase （PI3K） or reactive oxygen species （ROS） significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased. Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.

Biochemical association between the prevalence of genetic polymorphism and myocardial infarction

Genetic polymorphism has a vital role in the pathogenesis and development of myocardial infarction(MI).Single nucleotide polymorphism at any one of the amino acid sequences can result in a diseased state.A single gene can exhibit genetic polymorphism at more than one position giving rise to different variants.Genetic polymorphism of angiotensinogen(AGT)M235T,AGT T174M,and angiotensin-1-converting enzyme(ACE)I/D,endothelial nitric oxide synthase(eNOS),and methylenetetrahydrofolate reductase(MTHFR)can be a risk factor for MI.However,it is important to study the prevalence of genetic polymorphisms of these genes among different populations.MI is influenced by genetic polymorphism of various genes,including AGT,ACE,eNOS,MTHFR,etc.However,the association of genetic polymorphism of these genes varies among different populations,but different ethnic groups could show contradictory results.These genes have shown a positive association with risks of MI in some populations,whereas the results have not been consistent with every ethnic group.In this article,we have summarized the genetic variations in the aforementioned genes and their association with MI.

Remote ischemic preconditioning protects liver ischemia-reperfusion injury by regulating eNOS-NO pathway and liver microRNA expressions in fatty liver rats

BACKGROUND: Ischemic preconditioning (IPC) is a strategy to reduce ischemia-reperfusion (I/R) injury. The protective effect of remote ischemic preconditioning (RIPC) on liver I/R injury is not clear. This study aimed to investigate the roles of RIPC in liver I/R in fatty liver rats and the involvement of endothelial nitric oxide synthase-nitric oxide (eNOS-NO) pathway and microRNA expressions in this process. METHODS: A total of 32 fatty rats were randomly divided into the sham group, I/R group, RIPC group and RIPC+I/R group. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and nitric oxide (NO) were measured. Hematoxylin-eosin staining was used to observe histological changes of liver tissues, TUNEL to detect hepatocyte apoptosis, and immunohistochemistry assay to detect heat shock protein 70 (HSP70) expression. Western blotting was used to detect liver inducible NOS (iNOS) and eNOS protein levels and realtime quantitative polymerase chain reaction to detect miR-34a, miR-122 and miR-27b expressions. RESULTS: Compared with the sham and RIPC groups, serum ALT, AST and iNOS in liver tissue were significantly higher in other two groups, while serum NO and eNOS in liver tissue were lower, and varying degrees of edema, degeneration and inflammatory cell infiltration were found. Cell apoptosis number was slightly lower in the RIPC+I/R group than that in I/R group. Compared with the sham group, HSP70 expressions were significantly increased in other three groups (all P<0.05). Compared with the sham and RIPC groups, elevated miR-34a expressions were found in I/R and RIPC+I/R groups (P<0.05). MiR-122 and miR-27b were found significantly decreased in I/R and RIPC+I/R groups compared with the sham and RIPC groups (all P<0.05). CONCLUSION: RIPC can reduce fatty liver I/R injury by affecting the eNOS-NO pathway and liver microRNA expressions.

Inhibition of TGF-β1 by eNOS gene transfer provides cardiac protection after myocardial infarction

Objective： Endothelial nitric oxide synthase （eNOS） and nitric oxide （NO） have been implicated in protection against myocardial ischemia injury. This study was designed to explore a new method of therapy for myocardial injury by eNOS gene transfection. Methods： A rat model of myocardial infarction （MI） was established by left anterior descending （LAD） coronary artery ligation, eNOS gene in an adenovirus vector was delivered locally into the rat heart and hemodynamic parameters were examined after 3 weeks, Matrix metalloproteinase-2 and 9 （MMP-2, MMP-9） mRNA were measured by reverse transcription polymerase chain reaction （RT-PCR）, and the protein levels of eNOS, caspase-3, and transforming grouth factor 131 （TGF-131） were determined by western blot assay. Results： eNOS gene transfer significantly reduced cardiomyocyte apoptosis and improved cardiac function. In addition, eNOS significantly reduced the mRNA levels of MMP-2 and MMP-9. In the eNOS gene transfected group, the activation of caspase-3 and TGF-β1 were decreased. However, the protection was reversed by administration of the NOS inhibitor, N（o））-nitro-l-arginine methyl ester （L-NAME）. Conclusion： These results demonstrate that the eNOS provides cardiac protection after myocardial infarction injury through inhibition of cardiac apoptosis and collagen deposition, and suppression of TGF-β1.

Anticancer activity of Δ^(9)-tetrahydrocannabinol and cannabinol in vitro and in human lung cancer xenograft

Objective:To investigate the effects of Δ^(9)-tetrahydrocannabinol,the principal psychoactive compound of Cannabis sativa,and cannabinol,a Δ^(9)-tetrahydrocannabinol degradative product,on human non-small cell lung cancer cells.Methods:Δ^(9)-Tetrahydrocannabinol and cannabinol were tested for anticancer activity in human non-small cell lung cancer(A549)cells.The effects on cell proliferation,apoptosis,and phosphorylation profiles were examined.The effects of Δ^(9)-tetrahydrocannabinol and cannabinol on tumor growth were also investigated using a xenograft nude mouse model.Apoptosis and targeted phosphorylation were verified by immunohistochemistry.Results:Δ^(9)-Tetrahydrocannabinol and cannabinol significantly inhibited cell proliferation and increased the number of apoptotic cells in a concentration-dependent manner.The Δ^(9)-tetrahydrocannabinol-and cannabinol-treated cells had lower levels of phosphorylated protein kinase B[AKT(S473)],glycogen synthase kinase 3 alpha/beta,and endothelial nitric oxide synthase compared to the controls.The study of xenograft mice revealed that tumors treated with 15 mg/kg Δ^(9)-tetrahydrocannabinol or 40 mg/kg cannabinol were significantly smaller than those of the control mice.The tumor progression rates in mice treated with 15 mg/kg Δ^(9)-tetrahydrocannabinol or 40 mg/kg cannabinol were significantly slower than in the control group.Conclusions:These findings indicate that Δ^(9)-tetrahydrocannabinol and cannabinol inhibit lung cancer cell growth by inhibiting AKT and its signaling pathways,which include glycogen synthase kinase 3 alpha/beta and endothelial nitric oxide synthase.

Association between eNOS gene promoter polymorphism (-786T>C) and idiopathic recurrent pregnancy loss in Iranian women

Objective:To investigate the frequency of-786T>C variant in endothelial nitric oxide synthase(eNOS)gene promoter in Iranian women with recurrent pregnancy loss.Methods:Blood samples were obtained from 100 unrelated women affected by recurrent pregnancy loss and 100 unaffected women as the controls.Genomic DNA was extracted and-786T>C polymorphism in eNOS gene promoter was investigated by PCR-RFLP method.Statistical analyses and Hardy-Weinberg equilibrium in the groups of patients and controls were performed by Chi-square test and SPSS standard software(Version 21).Results:The frequency of homozygous TT was 40%in cases and 46%in the control group;the frequency of CC was 7%in cases and 5%in the control group;frequency heterozygote TC was 53%in cases and 49%in the control group.Genotype frequencies between the two groups showed no significant differences(P>0.05).Conclusions:The-786T>C polymorphism is not more frequent in recurrent pregnancy loss in this population.

Effect of Troglitazone on Expression of Adhesion Molecules and eNOS in Human Saphenous Vein Gaft

To investigate whether peroxisome proliferators-activated receptor-γ （PPARγ） ligand Troglitazone can reduce endothelial injury and activation during storage of harvested saphenous vein grafts. Segments of human saphenous vein graft were collected from 9 patients undergoing coronary bypass surgery and then divided into two equal parts of control and test specimens, were stored in ei- ther heparinized blood （control group） or heparinized blood containing 20 μmol/L troglitazone （test group） for 1 h at room temperature. Tissue distribution and protein expression of VCAM-I, ICAM-I, and endothelial nitric oxide synthase （eNOS） were compared using immunohistochemistry and Western blot analysis. Myeloperoxidase （MPO） activity, a marker of neutrophil sequestration in human saphenous vein grafts, was also measured in each group. The expression of ICAM-1 （753±132 versus 7201±934; P〈0.01） , VCAM-1 （3731±294 versus 8292±793; P〈0.01）, and MPO activity （1.52±0.42 U/g, 5.04±1.26 U/g P〈0.01） were significantly lower in test group. In contract, eNOS expression （7983±834 versus 3989±1008; P〈0.01） was significantly higher in test group. PPARγ ligand troglitazone might reduce endothelial injury during the storage period of human saphenous vein grafts.

Assessment of Reversibility in Pulmonary Hypertension Related to Congenital Heart Disease by Using Biomarkers and Clinical Features

Background:Reversibility of pulmonary hypertension(PH)is closely related to the treatment options for and prognosis of children with congenital heart disease.Objective:We combined patient-specific clinical features including diagnosis,age and echocardiographic results,and biomarkers of pulmonary vascular dysfunction to explore the noninvasive methods that can be used to accurately evaluate the reversibility of pulmonary hypertension in congenital heart disease(PH-CHD).Methods:Based on the preoperative systolic pulmonary arterial pressure(sPAP),70 CHD patients were divided into normal,PH-CHD suspected,and confirmed groups.Additionally,biomarkers of circulating endothelial cells(CECs),endothelin-1(ET-1),and endothelial nitric oxide synthase(eNOS)were detected.Patients were categorized into reversible(RPH)and irreversible(IRPH)groups according to the sPAP 6 months after surgery.Risk stratification was performed according to the clinical features and biomarkers.Results:CECs and ET-1 levels in the confirmed group were significantly higher.eNOS was higher in the confirmed and suspected groups than that in the normal group.CECs in the IRPH group were significantly higher compared to the RPH group.No such intergroup differences were observed with respect to ET-1 and eNOS levels.The ROC curve showed that the risk stratification was of high diagnostic value to evaluate reversibility.Conclusion:The CECs,eNOS,and ET-1 were closely related with PH-CHD.CECs and risk stratification have high practical value in assessing the reversibility of PH-CHD.

Too hard to die:Exercise training mediates specific and immediate SARS-CoV-2 protection

Several mechanisms may explain how exercise training mechanistically confers protection against coronavirus disease 2019(COVID-19).Here we propose two new perspectives through which cardiorespiratory fitness may protect against severe acute respiratory syndrome coronavirus-2(SARS-CoV-2).Physical exercise-activated adenosine monophosphate(AMP)-activated protein kinase(AMPK)signaling induces endothelial nitric oxide(NO)synthase(eNOS),increases NO bio-availability,and inhibits palmitoylation,leading to specific and immediate SARS-CoV-2 protection.AMPK signaling also induces angiotensin 1-7 release and enhances eNOS activation thus further mediating cardio-and renoprotection.Irisin,a myokine released from skeletal muscles during aerobic exercise,also participates in the AMPK/Akt-eNOS/NO pathway,protects mitochondrial functions in endothelial cells,and antagonizes renin angiotensin system proinflammatory action leading to reductions in genes associated with severe COVID-19 outcomes.Collectively,all the above findings point to the fact that increased AMPK and irisin activity through exercise training greatly benefits molecular processes that mediate specific,immediate,and delayed SARS-CoV-2 protection.Maintaining regular physical activity levels is a safe and affordable lifestyle strategy against the current and future pandemics and may also mitigate against obesity and cardiometabolic disease syndemics.Move more because a moving target is harder to kill.

High-density lipoprotein regulates angiogenesis by affecting autophagy via miRNA-181a-5p

We previously demonstrated that normal high-density lipoprotein(nHDL)can promote angiogenesis,whereas HDL from patients with coronary artery disease(d HDL)is dysfunctional and impairs angiogenesis.Autophagy plays a critical role in angiogenesis,and HDL regulates autophagy.However,it is unclear whether n HDL and d HDL regulate angiogenesis by affecting autophagy.Endothelial cells(ECs)were treated with n HDL and d HDL with or without an autophagy inhibitor.Autophagy,endothelial nitric oxide synthase(e NOS)expression,miRNA expression,nitric oxide(NO)production,superoxide anion(O2^(·-))generation,EC migration,and tube formation were evaluated.n HDL suppressed the expression of miR-181a-5p,which promotes autophagy and the expression of e NOS,resulting in NO production and the inhibition of O2^(·-)generation,and ultimately increasing in EC migration and tube formation.d HDL showed opposite effects compared to n HDL and ultimately inhibited EC migration and tube formation.We found that autophagy-related protein 5(ATG5)was a direct target of miR-181a-5p.ATG5 silencing or miR-181a-5p mimic inhibited n HDL-induced autophagy,e NOS expression,NO production,EC migration,tube formation,and enhanced O2^(·-)generation,whereas overexpression of ATG5 or miR-181a-5p inhibitor reversed the above effects of d HDL.ATG5 expression and angiogenesis were decreased in the ischemic lower limbs of hypercholesterolemic low-density lipoprotein receptor null(LDLr^(-/-))mice when compared to C57BL/6 mice.ATG5 overexpression improved angiogenesis in ischemic hypercholesterolemic LDLr^(-/-)mice.Taken together,nHDL was able to stimulate autophagy by suppressing miR-181a-5p,subsequently increasing e NOS expression,which generated NO and promoted angiogenesis.In contrast,d HDL inhibited angiogenesis,at least partially,by increasing miR-181a-5p expression,which decreased autophagy and e NOS expression,resulting in a decrease in NO production and an increase in O2^(·-)generation.Our findings reveal a novel mechanism by which HDL affects angiogenesis by regulating autophagy and provide a therapeutic target for d HDL-impaired angiogenesis.

Decreased and dysfunctional circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease

Background It has been widely demonstrated that endothelial progenitor cells are involved in several diseases and that they have therapeutic implications. In order to define the altered pulmonary vascular homeostasis in chronic obstructive pulmonary disease, we sought to observe the level and functions of circulating endothelial progenitor calls in patients with chronic obstructive pulmonary disease. Methods The total study population included 20 patients with chronic obstructive pulmonary disease and 20 control subjects. The number of circulating endothelial progenitor cells （CD34＋/CD133＋/IVEGFR-2＋cells） was counted by flow cytometry. Circulating endothelial progenitor cells were also cultured in vitro and characterized by uptake of Dil- acLDL, combining with UEA-I, and expression of von Willebrand factor and endothelial nitric oxide synthase. Adhesion, proliferation, production of nitric oxide, and expression of endothelial nitric oxide synthase and phosphorylated-endothelial nitric oxide synthase were detected to determine functions of circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease. Results The number of circulating endothelial progenitor cells in the chronic obstructive pulmonary disease group was lower than in the control group： （0.54±0.16）% vs. （1.15±0.57）%, P 〈0.05. About 80% of adherent peripheral blood mononuclear cells cultured in vitro were double labeled with Dil-acLDL and UEA-I. The 92% and 91% of them were positive for von Willebrand factor and endothelial nitric oxide synthase, respectively. Compared with the control, there were significantly fewer adhering endothelial progenitor cells in chronic obstructive pulmonary disease patients： 18.7±4.8/field vs. 45.0±5.9/field, P 〈0.05. The proliferation assay showed that the proliferative capacity of circulating endothelial progenitor cells from chronic obstructive pulmonary disease patients was significantly impaired： 0.135±0.038 vs. 0.224±0.042, P 〈0.05. Furthermore, nitric oxide synthase （112.06±10.00 vs. 135.41±5.38, P 〈0.05）, phosphorylated endothelial nitric oxide synthase protein expression （88.89±4.98 vs. 117.98±16.49, P 〈0.05） and nitric oxide production （（25.11±5.27） Iμmol/L vs. （37.72±7.10） μmol/L, P 〈0.05） were remarkably lower in endothelial cells from the chronic obstructive pulmonary disease group than the control. Conclusion Circulating endothelial progenitor cells were decreased and functionally impaired in patients with chronic obstructive pulmonary disease.