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.

NITRIC OXIDE SYNTHASE AND VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION IN HEPATOCELLULAR CARCINOMA AND THE CORRELATION WITH ANGIOGENESIS

Objective: To analyze the expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC) and its relation to angiogenesis. Methods: Tissue sections from 71 HCC patients were examined immunohistochemically for protein expression of iNOS, eNOS, and VEGF. Microvessal density (MVD) was counted by endothelial cells immunostained by anti-CD34 antibody. Results: Positive immunostaining for iNOS, eNOS was detected in 83.1% and 85.9% of HCC respectively. INOS and eNOS were not detected in normal hepatic tissue. MVD was 34.3±1.5/HP and 38.6±1.6/HP in HCC with positive staining for iNOS and VEGF while it was 31.2±2.8/HP, and 22.4±2.0/HP in HCC with negative staining for iNOS and VEGF (P<0.01). A correlation between NOS expression and VEGF in HCC was not observed. Conclusion: iNOS and eNOS may play a role in malignant transformation f post-hepatic cirrhosis. The expression of iNOS and VEGF favors angiogenesis of HCC.

Simvastatin Increases the Activity of Endothelial Nitric Oxide Synthase via Enhancing Phosphorylation

3-hydroxy-3-methylgulutaryl-coenzyme A （HMG-CoA） reductase inhibitors or statins are a kind of lipid-lowering agents and have been used for the prevention and treatment of Cardiovascular diseases. Recent studies suggested that statins, besides lowering cholesterol, may protect vessels by enhancing the activity of endothelial nitric oxide synthase （eNOS）. In the present study, we investigated if simvastatin increases eNOS activity through its phosphorylation in 293 cells （293-eNOS） with stable expression of eNOS. The results showed that incubation of 293-eNOS cells with simvastatin （10 μm/L） for 2 h significantly increased in the activity of eNOS as shown by the conversion of L-arginine to L-citrulline （2889.70±201.51 versus 5630.18＋218.75 pmol/min . mg proteins） （P〈0.01）. Western blotting revealed that simvastatin increased phosphorylation of eNOS at 1177 （ser） and also 495 （thr） but did not affect the overall expression of eNOS or inducible NOS. Further study found that simvastatin raised phosphorylation levels of Akt and AMPK, and such effect could be antagonized by Akt inhibitor or AMPK inhibitor. These results suggest that simvastatin could stimulate,the activity of eNOS via its phosphorylation by Akt and AMPK, which provides a new mechanism, other than lipid-lowering effect, for the cardiovascular protection of statins.

Expression and activity of inducible nitric oxide synthase and endothelial nitric oxide synthase correlate with ethanolinduced liver injury

AIM： To study the expression and activity of inducible nitric oxide synthase （iNOS） and endothelial nitric oxide synthase （eNOS） in rats with ethanol-induced liver injury and their relation with liver damage, activation of nuclear factor-KB （NF-κB） and tumor necrosis factor-α （TNF-α） expression in the liver. METHODS： Female Sprague-Dawley rats were given fish oil （0.5 mL） along with ethanol or isocaloric dextrose daily via gastrogavage for 4 or 6 wk. Liver injury was assessed using serum alanine aminotransferase （ALT） activity and pathological analysis. Liver malondialdehyde （MDA）, nitric oxide contents, iNOS and eNOS activity were determined. NF-κB p65, iNOS, eNOS and TNF-α protein or mRNA expression in the liver were detected by immunohistochemistry or reverse transcriptase-polymerase chain reaction （RT-PCR）. RESULTS： Chronic ethanol gavage for 4 wk caused steatosis, inflammation and necrosis in the liver, and elevated serum ALT activity. Prolonged ethanol administration （6 wk） enhanced the liver damage. These responses were accompanied with increased lipid peroxidation, NO contents, iNOS activity and reduced eNOS activity. NF-κB p65, iNOS and TNF-α protein or mRNA expression were markedly induced after chronic ethanol gavage, whereas eNOS mRNA expression remained unchanged. The enhanced iNOS activity and expression were positively correlated with the liver damage, especially the necro-inflammation, activation of NF-KB, and TNF-α mRNA expression. CONCLUSION： iNOS expression and activity are induced in the liver after chronic ethanol exposure in rats, which are correlated with the liver damage, especially the necro-inflammation, activation of NF-KB and TNF-α expression, eNOS activity is reduced, but its mRNA expression is not affected.

Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in association with neovascularization in human primary astrocytoma

Objective: To investigate the relationship between the expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and angiogenesis in primary astrocytoma. Methods: Thirty-seven primary astrocytomas and 4 astrocytic hyperplasia samples were collected and divided into three groups according to histological grade. The expression of eNOS, VEGF and factorⅧ related antigen (FⅧRAg) were assayed by immunohistochemistry. Microvascular density was assessed by FⅧRAg immunoreactivity. The intensity of immunoreactivity was graded according to the percentage of positive tumor cells. Results: No eNOS and VEGF were expressed in the astrocytes and vascular endothelium in astrocytic hyperplasia. The expression of eNOS or VEGF was light in low-grade astrocytoma and strong in glioblastoma. eNOS expression in astrocytoma was very positively correlated with VEGF. eNOS and VEGF expression in anaplastic astrocytoma was median in contrast to the low grade astrocytoma and glioblastoma. Lower microvascular density was found in low grade astrocytoma than that in higher grade malignant ones. The expressions of eNOS and VEGF were correlated with microvascular density and tumor malignancy. Conclusion: This finding suggests that eNOS and VEGF may have cooperative effect in tumor angiogenesis and play an important role in the pathogenesis of primary astrocytoma.

C1q/TNF-related protein 1 promotes vasodilatory dysfunctions by increasing arginase 1 activity and uncoupling of endothelial nitric oxide synthase

Objective C1q/TNF-related protein(CTRP)1 was initiallyidentified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins.Previously,we showed that CTRP1promotes the development of atherosclerosis by increasing endothelial adhesiveness.Here,we sought to investigate whether CTRP1 also influences vascular dilatory functions.

Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells

AIM： To study the expression of endothelial and inducible nitric oxide synthases （eNOS and iNOS） and their role in inflammatory bowel disease （IBD）. METHODS： We examined the effect of sera obtained from patients with active Crohn＇s disease （CD） and ulcerative colitis （UC） on the function and viability of human umbilical vein endothelial cells （HUVEC）. HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10^-4 mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. RESULTS： In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control （18%±16% vs 23%± 15% P〈0.01）. UC or CD serum caused a significant increase in iNOS compared to control （UC： 300%±21%; CD： 275% ± 27% vs 108% ± 14%, P〈0.01）. Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION： IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced antiinflammatory protection of endothelial cells.

Association between endothelial nitric oxide synthase(ENOS) G894T polymorphism and high altitude(HA) adaptation： a meta-analysis

Objective: Highland natives adapt well to the hypoxic environment at high altitude(HA). Several genes have been reported to be linked to HA adaptation. Previous studies showed that the endothelial nitric oxide synthase(ENOS) G894 T polymorphism contributed to the physiology and pathophysiology of humans at HA by regulating the production of NO. In this meta-analysis, we evaluate the association between the ENOS G894 T polymorphism and HA adaptation through analyzing the published data. Methods: We searched all relevant literature about the ENOS G894 T polymorphism and HA adaptation in Pub Med, Medline, and Embase before Step 2015. A random-effects model was applied(Revman 5.0), and study quality was assessed in duplicate. Six studies with 634 HA native cases and 621 low-altitude controls were included in this meta-analysis. Results: From the results, we observed that the wild-type allele G was significantly overrepresented in the HA groups(OR=1.85; 95% CI, 1.47–2.33; P<0.0001). In addition, the GG genotype was significantly associated with HA adaptation(OR=1.99; 95% CI, 1.54–2.57; P<0.0001). Conclusion: Our results showed that in 894 G allele carriers, the GG genotype might be a beneficial factor for HA adaptation through enhancing the level of NO. However, more studies were needed to confirm our findings due to the limited sample size.

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.

Changes in Human Umbilical Vein Endothelial Cells Induced by Endothelial Nitric Oxide Synthase Traffic Inducer

This study investigated the changes in human umbilical vein endothelial cells （HUVECs） induced by overexpression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the prolifera- tive ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS （eNOS） activity was assayed by using spectrophotometry, and NO2-/NO3- levels were measured usin~ nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma mem- brane and cytoplasm, and Western blot analysis confirmed that NOSTR1N levels were significantly higher in cells transfected with the NOSTR1N plasmid （P〈0.01）. The activity of eNOS and the levels of NO2-/NO3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells （P〈0.01 and P〈0.01, respectively）. Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOS- TRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overex- pression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.

Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase

Objective To find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase （eNOS）. Methods ＆ Results A major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin （BH4）. Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes： GTP cyclobydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin （BH2） as well as BH4：BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4： eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality （i.e., coupled or uncoupled）, the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable. Conclusions Although dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

Shear stress effect on endothelial nitric oxide synthase in cultured human umbilical vein endothelial cells

Background: Low shear stress caused by disturbed or turbulent flow at arterial branch points is known to associate with atherosclerosis. However, shear stress at the venous valve location and its association with deep vein thrombosis are less understood due to the complex and poorly understood bi-directional flow in the valve pocket region. We investigated how venous endothelial cells respond to flow shear stress around the venous valve region using a novel in vitro system that mimics venous flow. Results: Human umbilical vein EAhy. 926 cells were cultured on a flexible silastic membrane that mimicked venous tissue. Confluent cells were exposed to sinusoidal uni-and bi-directional pulsatile shear stress (0.1 to 1 dyne/cm2) for up to 6 h. Western-blot analyses indicated that endothelial nitric oxide (eNOS) expression levels decreased regardless of all tested flow patterns, stress magnitude, and shearing time. In contrast, the expression levels of inhibitor of κB (kappa B) and α (alpha)-tubulin were unaffected by the shear stress. Conclusions: Our results indicate that shear stress causes a decrease specifically in eNOS expression, suggesting that it may play a significant role in regulating inflammation related protein expression in endothelial cells.

Anti-cancer effect of iNOS inhibitor and its correlation with angiogenesis in gastric cancer

AIM: To observe the anti-cancer effect of iNOS selective inhibitor (aminoguanidine, AG) and investigate the relationship between iNOS inhibitor and angiogenesis, infiltration or metastasis in MFC gastric cancer xenografts.METHODS: Fifty athymic mice xenograft models were established by inoculating gastric cancer cell MFC subcutaneously. Twenty-four hours later, 0.9% sodium chloride solution, mitomycin, low dosage AG, high dosage AG, mitomycin and AG were administered by intraperitoneal injection respectively. Thus these mice were divided into five groups of 10 each randomly: control group, MMC group,AGL group, AGH group, MMC+AGH group. Two weeks later the mice were killed, and the tumor weight, inhibitory rate were evaluated. Greiss assay was used to detect the nitric oxide levels in plasma. HE and immunohistochemistry staining were used to examine microvessel density (MVD)and the expression of iNOS, VEGF, and PCNA. Apoptosis was detected by using TUNEL assay.RESULTS: The inhibitory rates in MMC+AGH group and AGH group were 52.9% and 47.1% respectively, which is significant statistically compared with that of control group (0). In treatment groups, the cell proliferation index (PI)was lower and apoptosis index was higher than those of control group. Microvessel density, iNOS, and VEGF in MMC+ AGH group were 8.8±2.6, 2.4±1.1, and 2.1±1.4respectively, which is significant statistically compared with those of control group (68.3±10.6, 11.3±1.3, and 10.3±1.6). The NO level in plasma of MMC+ AGH and AGH group were 12.7±2.1 and 12.9±2.0 μmol/L. Compared with that of control group (46.6±2.3 μmol/L), the difference is statistically significant.CONCLUSION: AG has anticancer effect on gastric cancer,and it has positive synergistic effect with chemotherapeutic drugs. It may play important inhibitory roles in angiogenesis of gastric cancer. The anticancer effect of iNOS inhibitors may include inducing cell apoptosis, suppressing cell proliferation and reducing angiogenesis.

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.

Neuronal nitric oxide synthase/reactive oxygen species pathway is involved in apoptosis and pyroptosis in epilepsy

Dysfunction of neuronal nitric oxide synthase contributes to neurotoxicity,which triggers cell death in various neuropathological diseases,including epilepsy.Studies have shown that inhibition of neuronal nitric oxide synthase activity increases the epilepsy threshold,that is,has an anticonvulsant effect.However,the exact role and potential mechanism of neuronal nitric oxide synthase in seizures are still unclear.In this study,we performed RNA sequencing,functional enrichment analysis,and weighted gene coexpression network analysis of the hippocampus of tremor rats,a rat model of genetic epilepsy.We found damaged hippocampal mitochondria and abnormal succinate dehydrogenase level and Na+-K+-ATPase activity.In addition,we used a pilocarpine-induced N2a cell model to mimic epileptic injury.After application of neuronal nitric oxide synthase inhibitor 7-nitroindazole,changes in malondialdehyde,lactate dehydrogenase and superoxide dismutase,which are associated with oxidative stress,were reversed,and the increase in reactive oxygen species level was reversed by 7-nitroindazole or reactive oxygen species inhibitor N-acetylcysteine.Application of 7-nitroindazole or N-acetylcysteine downregulated the expression of caspase-3 and cytochrome c and reversed the apoptosis of epileptic cells.Furthermore,7-nitroindazole or N-acetylcysteine downregulated the abnormally high expression of NLRP3,gasdermin-D,interleukin-1βand interleukin-18.This indicated that 7-nitroindazole and N-acetylcysteine each reversed epileptic cell death.Taken together,our findings suggest that the neuronal nitric oxide synthase/reactive oxygen species pathway is involved in pyroptosis of epileptic cells,and inhibiting neuronal nitric oxide synthase activity or its induced oxidative stress may play a neuroprotective role in epilepsy.

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.

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.

Relationships between endothelial nitric oxide synthase gene polymorphisms and osteoporosis in postmenopausal women

Objective： To investigate the relationships between endothelial nitric oxide synthases （eNOS） G894T and 27 bpvariable number tandem repeat （VNTR） gene polymorphisms and osteoporosis in the postmenopausal women of Chinese Han nationality. Methods： In the present study, 281 postmenopausal women from Xi＇an urban area in West China were recruited, and divided into osteoporosis, osteopenia, and normal groups according to the diagnostic criteria of osteoporosis proposed by World Health Organization （WHO）. The bone mineral density （BMD） values of lumbar vertebrae and left hips were determined by QDR-2000 dual energy X-ray absorptiometry. Blood samples were tested for plasma biochemical indicators including testosterone, estradiol, calcitonin, osteocalcin, and procollagen type I amino-terminal propeptide by enzyme-linked immunosorbent assay （ELISA）, tartrate-resistant acid phosphatase by spectrophotometric method, and the content of nitric oxide by Griess method. Genome DNA was extracted from whole blood, and G894T polymorphism of eNOS gene was analyzed by using polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） method and 27 bp-VNTR polymorphism of eNOS gene was genotyped by PCR method. Then the relationships between genotypes and biochemical indicators, genotypes and osteoporosis, and haplotypes and osteoporosis were analyzed. Results： The average BMD values of the femoral neck, ward＇s triangle and lumbar vertebrae 1-4 （LI-L4） in the subjects with T/T genotype in eNOS G894T locus were significantly higher than those in the subjects with G/T and G/G genotypes （P〈0.05）. The average BMD of the femoral neck in the subjects with a/a genotype of eNOS 27 bp-VNTR locus was evidently higher than that in the subjects with b/b genotype （P〈0.05）. The plasma testosterone and osteocalcin concentrations in the subjects of eNOS G894T G/T genotype were evidently higher than those in the subjects of other genotypes （P〈0.05）; the plasma estradiol concentration in the subjects of eNOS 27 bp-VNTR a/a genotype was obviously higher than that in the subjects of b/b genotype （P〈0.01）. eNOS G/G homozygous frequencies in osteoporosis women, osteopenia women, and normal women were 85.37%, 76.38%, and 83.87%, respectively （P〉0.05）. 0% osteoporosis woman, 0.79% osteopenia women, and 3.23% normal women were eNOS a/a homozygous （P〈0.05）. The frequencies of eNOS 27 bp-VNTR a allele were 5.33% in the osteoporosis group, 10.24% in the osteopenia group, and 16.13% in the normal group （P〈0.05, odds ratio （OR）=0.29, 95% confidence interval （CI）=0.11-0.77）, suggesting that a/a genotype and a allele might have protective effects on osteoporosis. The haplotype analysis showed that G-b was 87.7% （214/244） in the osteoporosis group （P〈0.05, OR=2.48, 95% CI=1. 18-5.18）. G-a was 5.3% （13/244） in the osteoporosis group （P〈0.05, OR=0.29, 95% CI=0. 11-0.77）. G-b was a risk factor for osteoporosis, and G-a a protective factor. Conclusion： eNOS G894T G/T genotype influenced the plasma testosterone and osteocalcin concentrations, and T/T genotype influenced BMD. eNOS 27 bp-VNTR a/a genotype increased plasma estradiol concentration to have a protective effect on osteoporosis.

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.

Endothelial Nitric Oxide Synthase Gene Polymorphisms Associated with Susceptibility to High Altitude Pulmonary Edema in Chinese Railway Construction Workers at Qinghai-Tibet over 4500 Meters above Sea Level

Objective To examine whether the polymorphisms of endothelial nitric oxide synthase (eNOS) gene are associated with the susceptibility to high altitude pulmonary edema (HAPE) in Chinese railway construction workers at Qinghai-Tibet where the altitude is over 4 500 m above sea level. Methods A case-control study was conducted including 149 HAPE patients in the construction workers and 160 healthy controls randomly recruited from their co-workers, matching the patients in ethnicity, age, sex, lifestyle, and working conditions. Three polymorphisms of eNOS gene, T-786C in promoter, 894G/T in exon 7, and 27bp variable number tandem repeat (VNTR) in intron 4, were genotyped using polymerase chain reaction (PCR) and confirmed with DNA sequencing. Results The frequencies of 894T allele and heterozygous G/T of the 894G/T variant were significantly higher in HAPE patients group than in the control group (P=0.0028 and P=0.0047, respectively). However, the frequencies of the T-786C in promoter and the 27bp VNTR in intron 4 were not significantly different between the two groups. Haplotypic analysis revealed that the frequencies of two haplotypes (H3,T-T-b, b indicates 5 repeats of 27 bp VNTR; H6, C-G-a, a indicates 4 repeats of 27 bp VNTR) were significantly higher in HAPE patients (both P<0.0001). On the contrary, the frequencies of H1 (T-G-b) and H2 (T-G-a) were lower in HAPE patients than in healthy controls (both P<0.001). Conclusions Two haplotypes (T-T-b and C-G-a) may be strongly associated with susceptibility to HAPE. Compared with the individual alleles of eNOS gene, the interaction of multiple genetic markers within a haplotype may be a major determinant for the susceptibility to HAPE.