Optimization of a synthetic beta-catenin-dependent promoter for tumor-specific cancer gene therapy.

We recently published the construction and evaluation of a beta-catenin-dependent, highly active promoter, CTP1, and its possible application for the treatment of colorectal cancer using gene-directed enzyme prodrug therapy with adenoviral (Ad) vectors. Alternative Ad-based approaches such as tumor-specific, replication-competent vectors and/or exploiting therapeutic gene products with intrinsic toxic activity, such as gibbon ape leukemia virus fusogenic membrane glycoprotein, diphtheria toxin A (DTA), and ricin, would demand a very tightly regulated promoter to avoid breakthrough replication and toxicity in nontumor tissue and Ad producer cell lines. In this study we optimized the activity/specificity profile of the synthetic beta-catenin-dependent promoter by varying its basal promoter, the number of Tcf binding sites, and the distance between these and the basal promoter. The optimal promoter, CTP4, showed virtually undetectable expression in cells with normal beta-catenin regulation but high level expression in cells deregulated for beta-catenin. Using CTP4 we were able to generate, for the first time to our knowledge, an Ad vector expressing fully active wild-type DTA without the need for time-consuming and cumbersome production systems. CTP4 should be the promoter of choice for Ad-based gene therapies of tumors deregulated for beta-catenin. We provide preliminary evidence that these may include prostate and ovarian as well as colorectal cancer.     

Development of a synthetic promoter for macrophage gene therapy

Macrophages have the potential to deliver therapeutic genes to many target tissues. Macrophage-specific synthetic promoters (SPs) generated by random ligation of myeloid/macrophage cis elements had activity up to 100-fold that of a native macrophage promoter in macrophage cell lines, but were minimally active in nonmyeloid cells. Mouse bone marrow cells (BMCs) transduced ex vivo with lentivectors expressing green fluorescent protein (GFP) driven either by an SP (SP-GFP) or a cytomegalovirus (CMV) promoter (CMV-GFP) were used for syngeneic transplantation of lethally irradiated mice. Blood leukocytes showed stable GFP expression for up to 15 months after transplantation. SP-GFP expression was selective for CD11b+ macrophages, whereas CMV-GFP expression was observed in erythrocytes, as well as in both CD11b+ and CD11b- leukocytes. Furthermore, SP-GFP expression was much stronger than CMV-GFP expression in CD11b+ macrophages. apoE-/- BMCs transduced with the lentiviral vector encoding human apoE were used to transplant apoE-/- mice. Macrophage expression of apoE from 10 to 26 weeks of age significantly reduced atherosclerotic lesions in recipient apoE-/- mice. Thus, the novel SPs, especially when combined with lentivectors, are useful for macrophage-specific delivery of therapeutic genes.     

诱导型一氧化氮合酶基因启动子区微卫星多态与急性冠状动脉综合征

目的:研究中国汉族人群诱导型一氧化氮合酶(NOS2A)基因启动子区-2.5kb微卫星CCTTT串联重复序列多态与急性冠状动脉综合征(ACS)的相关性。方法:应用微卫星分析技术,检测CCTTT微卫星多态在117例ACS患者和125名健康对照者中的频率分布。结果:根据CCTTT微卫星多态对NOS2A基因转录水平的影响,将CCTTT串联重复序列分为S(重复次数≤13)和L(重复次数>13)2类,基因型分布在ACS组(S/S=48,S/L=64,L/L=5)和对照组(S/S=56,S/L=49,L/L=20)间存在显著差异(χ2=11.354,P=0.003)。对照组的L/L基因型频率显著高于ACS组,r为0.234(95%CI=0.085～0.647,P=0.003),可能具有保护性作用。分层研究发现,女性ACS与对照组相比,基因型分布存在显著差异(χ2=8.134,P=0.017)。结论:NOS2A基因启动子区CCTTT微卫星多态可能与中国汉族ACS,尤其与女性患者的遗传易感性有关。     

Plasma nitric oxide concentrations and nitric oxide synthase gene polymorphisms in coronary artery disease

BACKGROUND: Plasma NOx (nitrate and nitrite) is a stable end product of the vasodilator NO. Several polymorphisms in the endothelial constitutive NO synthase (ecNOS) gene have been reported, including the 4a/4b VNTR polymorphism in intron 4, the E298D mutation in exon 7, and the G10-T polymorphism in intron 23. The aims of this study were to examine plasma NOx in patients with coronary artery disease (CAD) and to assess the association between plasma NOx concentrations and the three ecNOS gene polymorphisms. METHODS: Plasma NOx was measured in samples from 128 healthy controls and from 110 CAD patients at least 2 months after myocardial infarction. Three genetic polymorphisms that are known or have been suggested to be associated with plasma NOx concentration were also analyzed by PCR-restriction fragment length polymorphism. RESULTS: Median plasma NOx was significantly higher (P <0.001) in CAD patients (95.9 micromol/L) than in controls (73.8 micromol/L). Furthermore, the median plasma NOx was significantly higher (P <0.001) in hypertensive CAD patients (116.0 micromol/L) than in controls and normotensive CAD patients (86.0 micromol/L). The G-allele frequency of the G10-T polymorphism in intron 23 was significantly higher in CAD patients than in controls. Other polymorphisms showed no differences in allelic frequencies among the control and CAD groups. In controls, individuals with the E298D mutation in exon 7 (136.1 micromol/L) showed significantly higher (P = 0.001) median plasma NOx than those without this mutation (64.5 micromol/L). CONCLUSIONS: Plasma NOx was higher in hypertensive CAD patients than in normotensive CAD patients and controls. The E298D polymorphism of the ecNOS gene was associated with increased plasma NOx. Further study is needed to understand the gene expression and enzyme activity of ecNOS and their association with genotypes.     

In vitro modulation of inducible nitric oxide synthase gene expression and nitric oxide synthesis by procalcitonin

OBJECTIVE: Serum procalcitonin (PCT) concentration was recently introduced as valuable diagnostic marker for systemic bacterial infection and sepsis. At present, the cellular sources and biological properties of PCT are unclear. During sepsis and septic shock, inducible nitric oxide synthase (iNOS) gene expression is stimulated followed by the release of large amounts of nitric oxide (NO). We investigated the possible association between PCT and iNOS gene expression in an in vitro cell culture model. DESIGN: Prospective, controlled in vitro cell culture study. SETTING: University research laboratories. INTERVENTIONS: Confluent rat vascular smooth muscle cells (VSMC) were incubated for 24 hrs and 48 hrs with PCT (1 ng/mL, 10 ng/mL, 100 ng/mL, 1,000 ng/mL, 5,000 ng/mL) alone or with the combination of tumor necrosis factor-alpha (TNF-alpha, 500 U/mL) plus interferon-gamma (IFN-gamma, 100 U/mL). iNOS gene expression was measured by qualitative as well as quantitative polymerase chain reaction analysis, NO release was estimated by the modified Griess method. MEASUREMENTS AND MAIN RESULTS: PCT in increasing concentrations had no effect on iNOS gene expression and nitrite/nitrate release for 24 hrs and 48 hrs, respectively. However, PCT ameliorated TNF-alpha/IFN-gamma-induced iNOS gene expression in a dose-dependent manner (maximal inhibition at PCT 100 ng/mL by -66% for 24 hrs and -80% for 48 hrs). This was accompanied by a significantly reduced release of nitrite/nitrate into the cell culture supernatant (maximal reduction at PCT 100 ng/mL by -56% and -45% for 24 hrs and 48 hrs, respectively). CONCLUSIONS: We conclude that recombinant PCT inhibits the iNOS-inducing effects of the proinflammatory cytokines TNF-alpha/ IFN-gamma in a dose-dependent manner. This might be a counter-regulatory mechanism directed against the large production of NO and the concomitant systemic hypotension in severe sepsis and septic shock.     

Preclinical evaluation of inducible nitric oxide synthase lipoplex gene therapy for inhibition of stent-induced vascular neointimal lesion formation

Several reports have established the concept of nitric oxide synthase (NOS) gene transfer for inhibiting smooth muscle cell (SMC) proliferation after vascular injury. To minimize potential risks associated with viral gene transfer, we developed a liposome-based gene transfer approach employing inducible NOS (iNOS) overexpression for inhibition of stent-induced neointimal lesion formation. Therapeutic lipoplexes were transferred to femoral or coronary arteries of Goettingen minipigs, using the Infiltrator local drug delivery device. Efficiency of local iNOS lipoplex transfer was analyzed by iNOS-specific immunohistochemistry. NO-mediated inhibition of stent-induced neointimal lesion formation was analyzed by intravascular ultrasound (IVUS) and computerized morphometry. Gene transfer efficiency increased dose dependently to a maximum of 44.3 +/- 4.2% iNOS-positive vessel area (dose, 2 microg of iNOS lipoplex). Proliferating cell nuclear antigen (PCNA) expression of medial SMCs (immunohistochemistry) was inhibited significantly by transfer of 2 microg of iNOS lipoplexes (111 +/- 27 cells [iNOS] versus 481 +/- 67 cells [control; PCNA-positive medial cells]). IVUS analysis demonstrated that local transfer of iNOS lipoplexes resulted in a significant reduction of femoral in-stent plaque area (control, 40.85 +/- 6.37 mm(2); iNOS, 24.69 +/- 1.8 mm(2); p = 0.03). Coronary in-stent lesion formation was reduced by about 45% as determined by histologic morphometry (control, 4.0 +/- 0.29; iNOS, 2.2 +/- 0.30; p < 0.01). In conclusion, this study demonstrates that local intramural delivery of iNOS lipoplexes can exert therapeutic effects in inhibiting stent-induced neointimal lesion formation. Together with the nonviral character of this gene therapy approach, these findings may have important impact on the transition of NOS-based gene therapy to clinical practice.     

Development of a prostate-specific promoter for gene therapy against androgen-independent prostate cancer

Androgen ablation has been the standard treatment for metastasized prostate cancer. In most cases, however, prostate cancer cells eventually lose androgen dependency and become refractory to the conventional endocrine therapy. Androgen-independent prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cells. Prostate-specific promoters such as prostate-specific antigen and rat probasin (rPB) promoters have been examined in the development of gene therapy targeted to prostate cancer. However, those promoters require binding of the androgen–AR complex to the androgen-response element and are active only in the androgen-dependent prostate cancer cell lines and not in the androgen-independent cell lines. To target transgene expression in androgen-independent prostate cancer, we designed a prostate-specific promoter that is activated by the retinoids–retinoid receptor complex instead of the androgen–AR complex. The modified rPB promoters expressed transgenes in response to retinoid in both androgen-dependent and androgen-independent prostate cancer cells and not in other cancer cell lines or in human normal cells, in vitro and in vivo. Furthermore, the combination of retinoid treatment and adenovirus-mediated gene transfer of the modified rPB-driven HSV-tk gene resulted in a significant growth suppression of the androgen-independent prostate cancer cells in the presence of the prodrug ganciclovir. This study suggests that tailoring of the hormone-responsive elements may offer a new therapeutic opportunity against the hormone-refractory stage of prostate cancer.     

内皮一氧化氮合酶基因多态性与糖尿病肾病关系的研究

目的 探讨内皮一氧化氮合酶(eNOS)基因内含子4的多态性与糖尿病肾病的关系,以及其在中国人、马来西亚人和印度人中的分布。方法 选择258例病程在10年以上的2型糖尿病患者作为观察对象,其中中国人150例、马来西亚人71例、印度人37例。从患者全血中提取DNA,然后,进行聚酶链反应(PCR)、克隆及测序。结果 以往认为该基因有2种等位基因(a和b),而本研究发现3种等位基因,并对第3种多态(等位基因c)基因进行了序列分析;统计分析显示,等位基因a、b和c均与糖尿病肾病无显著相关性。结论 eNOS基因内含子4上的多态性有3种等位基因;该基因多态性与糖尿病肾病无显著相关性。     

Regulation of nitric oxide synthase]

Nitric oxide (NO) synthase is known to be widely distributed in various cells. We characterized this enzyme using partially purified enzyme fractions from rat neutrophils, macrophages, and cerebellum. The cerebellar fraction required Ca(2+)-calmodulin, while that from macrophages required neither calmodulin nor Ca2+. The neutrophil fraction required Ca2+. The enzyme was inhibited by analogues of the substrate, L-arginine. N omega-nitro-L-arginine (NNA) was 20 times more potent than L-NG-monomethyl-arginine (NMA) in blocking the cerebellar enzyme. In contrast, NNA and NMA were about equipotent against neutrophil and macrophage enzymes. These data suggest that the enzyme is regulated by 3 different mechanisms in these cells, with differences between the cerebellar and neutrophil or macrophage enzyme in the catalytic binding site.     

大鼠小肠移植术后血清一氧化氮及小肠组织一氧化氮合酶和诱导型一氧化氮合酶活性的变化

目的:研究同种大鼠小肠移植后内源性一氧化氮、一氧化氮合酶(nitricoxidesynthase,NOS)及诱导型一氧化氮合酶(induciblenitricoxidesyn-thase,iNOS)的变化及一氧化氮与急性排斥反应的关系。方法:以大鼠小肠移植为研究对象,16只SD大鼠进行同系移植作为对照组,8只SD大鼠和8只Wistar大鼠进行同种移植作为实验组,两组移植后分别于第3,5,7天同时取血液及小肠组织,病理为常规苏木精-伊红染色观察,血清一氧化氮采用硝酸还原酶法测定,NOS和iNOS采用分光光度法测定。结果:在急性排斥反应发生的早期实验组血清一氧化氮水平与对照组比较显著升高(术后第3,5,7天t值分别为9.7900,9.0073,6.3159,P<0.01),小肠组织NOS及iNOS活性亦显著高于对照组(NOS术后第3,5,7天t值分别为5.9318,9.1237,3.0457,iNOS术后第3,5,7天t值分别为3.2008,5.4930,4.8170,P<0.01)。结论:大鼠小肠移植后NOS及iNOS变化与排斥反应相关,血清一氧化氮水平的检测可作为干预移植措施始动的指标之一。     

Nitric oxide synthase gene therapy: progress and prospects

NOS gene therapy has been the focus of extensive research as dysfunction of this enzyme has been implicated in several cardiovascular diseases. Research has concentrated on comparing the effect of gene delivery of NOS isoforms (eNOS, iNOS and nNOS) in healthy and diseased animal models on intimal hyperplasia, restenosis, vascular tone and ischemia-reperfusion injury. Most results demonstrate therapeutic benefits following vascular gene delivery of all NOS in pre-clinical models of cardiovascular disease. eNOS has been shown to have particular promise as it promotes re-endothelialisation and inhibits intimal hyperplasia in injured blood vessels. The ultimate goal is to translate the benefit of NOS gene therapy in animal models into clinical practise. To develop NOS gene therapy for clinical use further work needs to be undertaken to improve delivery systems and vectors to minimise detrimental side-effects and enhance positive treatment outcomes. This review focuses on current research on NOS gene therapy in cardiovascular disease and identifies the next steps that would be necessary to lead to clinical trials.     

人内皮型一氧化氮合成酶基因转染桡动脉的表达

目的:桡动脉作为血管桥材料在冠状动脉旁路移植术中具有重要作用,但血管桥痉挛一直是困扰桡动脉移植的难题.实验通过腺病毒载体介导的人内皮型一氧化氮合成酶基因(AdCMVeNOS)转染人桡动脉,体外培养后检测人内皮型一氧化氮合成酶基因的表达,观察其对内膜增生的影响.方法:①实验于2005-03104在解放军沈阳军区总医院完成.实验用Enos-Ad由该院麻醉科张铁铮主任惠赠.桡动脉来自15例冠状动脉旁路移植手术患者剩余桡动脉.将桡动脉横切成5 mm血管环,采用随机数字表法分为3组,对照组、空载腺病毒液感染组、内皮型一氧化氮合酶基因转染组,每组8份.空载腺病毒液感染组、内皮型一氧化氮合酶基因转染组桡动脉血管环分别置于空载腺病毒液和AdCMVeNOS溶液中1 h,取出后体外培养14 d.应用多聚寡核苷酸探针和高敏感标记技术检测外源性基因内皮型一氧化氮合酶mRNA的表达:免疫组织化学染色方法检测内皮型一氧化氮合酶蛋白的表达:苏木精-伊红染色及弹性纤维染色后,应用计算机图像分析系统检测桡动脉内膜、中膜增生情况.结果:①3组培养的桡动脉在14 d后有新内膜形成和显著的中膜增厚,内皮型一氧化氮合酶基因转染组内膜厚度及内膜/中膜厚度比值低于对照组(P＜0.01),空载腺病毒液感染组与对照组差异无显著性(P＞0.05).②培养14d后,内皮型一氧化氮合酶基因转染组内膜和中膜可检测到内皮型一氧化氮合酶mRNA和蛋白质表达,对照组及空载腺病毒液感染组未见明显表达.结论:腺病毒载体介导的人内皮型一氧化氮合成酶基因成功转染体外培养桡动脉中并有效表达,内皮型一氧化氮合酶基因具有防治体外培养桡动脉内膜增生的作用.     

Modulation of nitric oxide synthase by nitric oxide donor compounds in migraine

A controlled study was performed to assess the involvement of the nitric oxide pathway in migraine pathophysiology. Thirteen patients with migraine without aura and seven clinically healthy subjects (C) were selected. All of the migraine patients were studied both before, during an asymptomatic phase (t ₀), and 1 h after the administration of 5 mg isosorbide dinitrate, a nitric oxide donor able to induce an experimental migraine attack (t ₁). The nitric oxide levels were analyzed as nitrite accumulation in serum samples, in peripheral blood mononuclear cell extracts, and culture supernatants. Basal nitrite levels in serum samples and peripheral blood mononuclear cell culture supernatants of migraine patients and healthy subjects indicated that migraine patients possess an activated nitric oxide synthesis pathway (t ₀ vs. C F=8.16,P<0.01 and F=16.2,P<0.01, respectively). As expected, in the migraine patients treated with the nitric oxide donor, a marked increase of nitrite levels was observed in sera (t ₁ vs.t ₀ P<0.05,t=3.05). In contrast, during the nitric oxide donor-induced migraine attacks a statistically significant decrease of nitrite levels in peripheral blood mononuclear cell culture supernatants was observed (t ₁ vs.t ₀ P<0.01,t=−4.03), whereas a significant increase of nitrite in total cell extracts was detected (t ₁ vs.t ₀ P<0.001,t=−6.89). These preliminary data suggest that nitric oxide could be involved in the neurovascular modifications leading to a migraine attack.     

Endothelial nitric oxide synthase gene polymorphisms and risk of coronary artery disease

BACKGROUND: Endothelial nitric oxide synthase (eNOS) could be a candidate gene for coronary artery disease (CAD). This study investigated the relationship of the eNOS Glu(298)-->Asp and T(786)-->C polymorphisms with the presence and severity of CAD in the Italian population. METHODS: We enrolled 415 unrelated individuals who underwent coronary angiography. The severity of CAD was expressed by means of the Duke score. The eNOS Glu(298)-->Asp and T(786)-->C variants were analyzed by PCR. RESULTS: There was significant linkage disequilibrium between the two eNOS polymorphisms (P <0.0001). Both variants were significantly associated with the occurrence and severity of CAD (P = 0.01 and 0.004 for Glu(298)-->Asp and T(786)-->C, respectively). The risk of CAD was increased among individuals homozygous for the C allele of the T(786)-->C polymorphism compared with individuals homozygous for the T allele (odds ratio = 2.5; P <0.01) and was independent of the other common risk factors (P = 0.04). Moreover, individuals with both the Asp/Asp genotype of the Glu(298)-->Asp polymorphism and at least one C allele of the T(786)-->C variant in the promoter region of the eNOS gene had an increased risk of CAD (odds ratio = 4.0; P <0.001) and a significantly higher mean Duke score (26.2 +/- 2.9 vs 45.2 +/- 3.7; P = 0.002) compared with individuals with the TT genotype and the Glu allele. CONCLUSIONS: The present study provides evidence that the Glu(298)-->Asp and T(786)-->C polymorphisms of the eNOS gene are associated with the presence and severity of angiographically defined CAD in the Italian population and that those individuals carrying both eNOS variants simultaneously might have a higher risk of developing CAD.     

Endogenously generated nitric oxide by nitric-oxide synthase gene transfer inhibits cellular proliferation

We investigated whether endothelial nitrite oxide synthase (NOS) gene transfer inhibited cellular proliferation. Endothelial NOS and endothelin type A receptor genes were transferred into 293 cells, a human embryonic kidney cell line, by calcium-phosphate coprecipitation. The cytosolic free Ca(2+) levels ([Ca(2+)](i)) of transfected cells were estimated with fura-2 fluorescence. Thymidine incorporation was increased by endothelin-1 in type A receptor-transfected cells. The endothelial NOS gene transfer did not affect endothelin-1-induced increase in [Ca(2+)](i) of type A receptor-transfected cells, but markedly inhibited mitogen-activated protein kinase and c-fos promoter activities. The endothelial NOS gene transfer also inhibited thymidine incorporation into type A receptor-transfected cells in response to endothelin-1, which was abolished in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine acetate. The endothelin-1-induced increase in cell number was significantly suppressed by endothelial NOS gene transfer as well as by the mitogen-activated protein kinase inhibitor PD98059. These results indicate that endothelial NOS gene transfer inhibits cellular proliferation via inhibition of the mitogen-activated protein kinase cascade.