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.

Identification and Pathogen Stimulation Patterns of Neuronal Nitric Oxide Synthase(nNOS)in Black Rockfish(Sebastes schlegelii)

Neuronal nitric oxide synthase(nNOS)was the producer of nitric oxide(NO)which played important gas messenger molecules in biological process.It also can take effect as immune regulation molecule in organism.Black rockfish(Sebastes schlegelii)is an important economic fish which were widely farmed in East Asia countries.Meanwhile,the pathogenic bacteria such as the Edwardsiella tarda and Vibrio anguillarum in seawater always brought serious obstacles to their healthy growth.In order to explore the expression pattern of n NOS gene under the pathogen stimulation and predict its immune function,the n NOS gene in black rockfish named Ssn NOS was identified.It was 3780 bp in length,located on chromosome 6,and contained 27 coding domain sequence(CDs).According to the phylogenetic analysis,the Ssn NOS showed closest relative to the counterpart gene of swamp eel(Monopterus albus).Meanwhile,analysis of Ssn NOS expression in various healthy tissues showed that Ssn NOS expression level was highest in healthy brain tissues,followed by intestinal tissues.In addition,Ssn NOS showed significant expression changes in response to stimulation by two pathogens.Particular in gill,the expression of Ssn NOS after pathogenic stimulation increased significantly.The Elisa analysis showed the Ssn NOS content in gills was much higher than that in other tissues at all time points.Moreover,the expression patterns of Ssn NOS in brain,intestine and kidney after stimulation by pathogens showed a distinct expression pattern which first down-regulated and then up-regulated.Therefore,the Ssn NOS may be an important signaling molecule for fish to respond rapidly in immune stimulation.

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.

Regeneration of neuronal nitric oxide synthase (nNOS)-containing nerve fibers in rat corpus cavernosum

Aim: To investigate the effect of cavernous nerve injury on the nNOS-containing nerve fibers in rat corpus cavernosum.Methods: Thirty-three male SD rats were randomized into 3 groups: 5 rats underwent pelvic exploration without tran-section of cavernous nerve as the sham-operated controls, the unilateral injury group (14 rats) had the cavernous nerve cuton one side, and the bilateral injury group (14 rats) had the nerves cut on both sides. Corpora cavernosa were harvestedat the 3rd week and 6th month after surgery, nNOS-positive nerve fibers were examined with strepavidin peroxidase im-munohistochemistry techniques (SP method). Results: After bilateral ablation, the nNOS-positive nerve fibers weresignificantly decreased at both the 3rd week ( 17 ± 4) and the 6th month (16 ± 4). For the unilateral injury group, thenNOS-positive nerve fibers were similarly decreased on the side of the neurotomy at the 3rd week (18 ± 6), but by the 6thmonth, the number increased significantly (61±9) and approximated the level on the contralateral side (81 ± 13). Con-clusion: In rats after unilateral cavernous nerve ablation, nNOS-containing nerve fibers might regenerate 6 months afteroperation, but regeneration did not occur in animals with bilateral cavernous nerve injury. Results suggest that duringpelvic radical surgery, the cavernous nerve should be preserved at least on one side in order to accomplish adequate regen-eration. (Asian J Androl 1999 Sep ; 1: 135 - 138)

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.

Effect of Berberine on the mRNA Expression of Nitric Oxide Synthase(NOS) in Rat Corpus Cavernosum

Summary: In order to further investigate the mechanisms of action of berberine (Ber), we assessed the effects of Ber on the mRNA expression of nitric oxide synthases (NOS) in rat corpus cavernosum. After incubation with Ber for 1 or 3 h respectively, the levels of NOS mRNA were examined by reverse transcription polymerase chain reaction (RT-PCR). Our results showed that there were iNOS and eNOS mRNA expressions in rat corpus cavernosum. Ber enhanced eNOS mRNA expression in rat penis, but exhibited no effect on the expression of iNOS mRNA (P>0.05). The present study indicated that the relaxation of Ber involved the NO-cGMP signal thansduction pathway. The enhancing effect of Ber on eNOS mRNA expression might associated with its relaxation of corpus cavernosum.

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.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma:Correlation with microvessel density

AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.

Expressions of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma

AIM： To determine the expressions of inducible nitric oxide synthase （iNOS） and matrix metalloproteinase-9 （MMP-9） in hepatocellular carcinoma （HCC） and to investigate the relationship between iNOS and MMP-9 expression and their effects on angiogenesis and progression of HCC.METHODS： In this study, we examined iNOS, MMP-9, and CD34 expression in specimens surgically removed from 32 HCC patients and 7 normal liver tissues by immunohistochemical staining. Meanwhile, microvessel density （MVD） was determined as a marker of angiogenesis by counting CD34-positive cells. RESULTS： The positive rates of iNOS and MMP-9 expression were 71.88% （23/32） and 78.13% （25/32） in HCC. MMP-9 expression was significantly correlated with tumor size, capsule status, TNM stage, and risk of HCC recurrence （P = 0.032, P= 0.033, P= 0.007, and P= 0.001, respectively）. There was also a significant relationship between iNOS expression and capsule status and risk of HCC recurrence （P = 0.049 and P = 0.004, respectively）, but no correlation between iNOS expression and tumor size and TNM stage. There was a positive association between MVD and TNM stage and risk of HCC recurrence （P = 0.037 and P = 0.000, respectively）. The count of MVD was significantly different in different iNOS and MMP-9 immunoreactivity groups （F= 17.713 and 17.097, P= 0.000 and P = 0.000, respectively）. The examination of Spearman＇s rank correlation coefficient showed that there was a significant positive correlation between MVD and iNOS, MMP-9 immunoreactivity （r = 0.754 and 0.751, P= 0.000 and P=-0.000, respectively）. There was also a significant association between MMP-9 and iNOS expression in HCC （P = 0.010）. CONCLUSION： Nitric oxide （NO） produced by iNOS could modulate MMP-9 production and therefore contribute totumor cell angiogenesis and invasion and metastasis in HCC. The strong expression of iNOS and MMP-9 in HCC may be helpful in evaluating the recurrence of HCC, predicting poor prognosis. For patients with strong expression of MMP-9 and iNOS, the optimal treatment scheme needs to be selected.

Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA

AIM: To investigate the expression of NOS in gastric carcinoma, and to explore the relationship between the expression of nitric oxide synthases (NOS) and p53, PCNA,pathological features and clinical staging of gastric cancer.METHODS: The activity of NOS protein was investigated in 85 samples of human gastric carcinoma and 25 samples of normal gastric mucosal tissue by biochemical assay. We then examined the expression of NOS, p53, PCNA in 85 samples of human gastric cancer was examined by immunohistochemistry, and NOS mRNA expression in 85 gastric cancer tissue specimens by in situ hybridization.RESULTS: Biochemical assay showed that the activity of NOS was significantly higher in gastric carcinoma than in normal gastric mucosal tissues (t = 0.4161, P＜0.01).Immunohistochemistry revealed that endothelial nitric oxide synthase (eNOS) expressed in all samples of normal gastric mucosa, but only 6 cases of 85 gastric cancer specimens showed weak positive immunohistochemical reactions to eNOS (20%). Inducible nitric oxide synthase (iNOS) was expressed strongly in human gastric carcinoma (81.2%). In situ hybridization analysis showed that iNOS mRNA expression was significantly stronger than eNOS mRNA expression in gastric cancer tissue (x2 = 10.23, P＜0.01). The expression of iNOS in gastric cancer was associated with differentiation, clinical stages or lymph node metastases (r= 0.3426, P＜0.05). However,iNOS expression did not correlate with histological classifications and morphological types. The expression of iNOS was significantly correlated with p53 or PCNA expression (r = 0.3612, P＜0.05). The expression of neuronal nitric oxide synthase (nNOS) was not examined by immunohistochemistry and in situ hybridization in gastric cancer specimens and normal gastric mucosa.CONCLUSION: In human gastric cancer, there is an enhanced expression of iNOS, but not of eNOS. NOS promotes the proliferation of tumor cells and plays an important role in gastric cancer spread. Inactivation of antioncogene p53 and overexpression of iNOS might play a synergetic role in the process of carcinogenesis of human gastric carcinoma.

Inducible nitric oxide synthase expression is related to angiogenesis,bcl—2 and cell proliferation in hepatocellular carcinoma

In this study, we examined the expression of inducible nitric oxide s ynthase (iNOS) and vascular endothelial growth factor (VEGF) by immunohistoc hemi cal staining in 76 tissue sections collected from hepatocellular carcinoma (HCC) patients undergoing hepatectomy. Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody. We performe d DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expressi o n on the cell cycle of HCC. Most of the HCC cells that invaded stroma were mark edly immunostained by iNOS antibody. The iNOS stain intensity of the liver tissu e close to the tumor edge was stronger than that of HCC tissue, and the stronges t was the hepatocytes closer to the tumor tissue. However, iNOS expression in 10 normal hepatic samples was undetectable. VEGF positive expression ratio was 84. 8% in iNOS positive expression cases, and the ratio was 35.3% in negative cases. There was significant correlation (P=0.000) between iNOS and VEGF expressi on. Moreover, iNOS expression was significantly associated with bcl-2 and MVD, but w ithout p53 expression. DNA-flow cytometric analyses showed that combined expres s ion of iNOS and VEGF had significant impact on the cell cycle in HCC. PI (Proli ferating Index) and SPF (S-phase fraction) in the combined positive expression o f iNOS and VEGF group was significantly higher than that in the combined negativ e group. The present findings suggested that iNOS expression was significantly a ssociated with angiogenesis, bcl-2 and cell proliferation of HCC.

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.

Differential expression of apoptosis related proteins and nitric oxide synthases in Epstein Barr associated gastric carcinomas

AIM： To determine the incidence of Epstein Barr virus associated gastric carcinoma （GC） in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negative gastric carcinoma. METHODS： In situ hybridization of EBV-encoded small RNA-1 （EBER-1） and PCR was performed to identify the presence of EBV in GCs. Immunohistochemistry was used to identify expressions of bcl-2, bcl-xl, bak, bax, p53, NOS-1, NOS-2, and NOS-3 proteins in 25 EBV positive GCs and in 103 EBV negative GCS. RESULTS： 12% of the cases of GC （25/208） showed EBER-1 and EBNA-1 expression. The cases were preferentially of diffuse type with intense lymphoid infiltrate in the stroma. EBV associated GCs showed higher expression of bcl-2 protein and lower expression of bak protein than in EBV negative GCs. Indeed, expressions of NOS-1 and NOS-3 were frequently observed in EBV associated GCs. CONCLUSION： Our data suggest that EBV infection may protect tumor cells from apoptosis, giving them the capacity for permanent cell cycling and proliferation. In addition, EBV positive GCs show high expression of constitutive NOS that could influence tumor progression and aggressiveness.

Synthesis of novel methotrexate derivatives with inhibition activity of nitric oxide synthase

Seventeen 4-alkylamino/arylamino-substituted methotrexate （MTX） derivatives 6a-14a were designed and synthesized. Their inhibition activities against inducible nitric oxide synthase （iNOS） were evaluated in vitro. The pharmacological results showed that most of the prepared compounds displayed the potent inhibitory effects on iNOS.

Role of Nitric Oxide and Nitric Oxide Synthases in Ischemia-reperfusion Injury in Rat Organotypic Hippocampus Slice

To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthasc isoforms （nNOS and iNOS）, rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen glucose deprivation （OGD） for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD （P〈0.05）. The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase （LDH） efflux into the incubation solution. The resuits suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.

Studies on the Compounds of d4T Combined with Nitric Oxide Donors and Nitric Oxide Synthase Inhibitors and their Anti-HIV and AIDS Activity

Stavudine, a potent anti HIV and AIDS related complex, is one of the Nucleoside Analogue Reverse Transcriptase Inhibitors (NARTIs). It is phosphorylated intracellularly and then inhibits the viral reverse transcriptase by acting as a false substrate. Modifications made on the hydrogen labile at the 5' position on the sugar is an interesting template for the elaboration of new potent anti HIV and AIDS drugs. The expected advantages of the modified stavudine prodrugs can be multiple: synergistic drug activities, enhancement of stavudine intracellular uptake, increase of stavudine brain delivery, and bypass of the first stavudine phosphorylation step into the cells. Nitric oxide synthase inhibitors of stavudine and nitric oxide donors of stavudine may hold significant promise for the treatment of HIV and AIDS.

Synthesis and biological evaluation of 2,4-diaminopteridine derivatives as nitric oxide synthase inhibitor

A series of novel 2,4-diamino-pteridines （9a-l） were synthesized and evaluated as inhibitors of inducible nitric oxide synthase （iNOS） in vitro. It was found that 9a, 9d, 9e, 9h, 9i and 91 showed potent inhibitory activities similar to that of methotrexate （MTX）, while the activities of 9b, 9c, 9f, 9g, 9j and 9k are stronger than MTX.

Potential involvement of nitric oxide synthase but not inducible nitric oxide synthase in the development of experimental corneal neovascularization

AIM: To investigate the effect of nitric oxide and its synthetase on experimental corneal neovascularization (CRNV). METHODS: CRNV was induced by alkali injury in mice, nitric oxide synthetase (NOS) was inhibited by NG-nitro-L-arginine (L-NAME) and inducible nitric oxide synthetase (iNOS) was inhibited by aminoguanidine hemisulfate salt (AG). The inhibitory effect was detected at day 2 and 4 after corneal alkali injury by reverse transcription polymerase chain reaction (RT-PCR). CRNV was compared between the control and the treated mice by microscopic observation and corneal whole mount CD31 immunostaining. RESULTS: The inhibition of L-NAME to NOS and AG to iNOS after corneal injury was confirmed by RT-PCR (P <0.05). Compared with control mice, L-NAME treated mice exhibited significantly decreased CRNV areas (P<0.05). In contrast, AG treatment failed to attenuate alkali induced CRNV (P>0.05). CONCLUSION: Our findings suggest that NOS but not iNOS plays a critical role in alkali injury induced CRNV.

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.

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.