Aspirin inhibits inducible nitric oxide synthase expression and tumour necrosis factor-alpha release by cultured smooth muscle cells

BACKGROUND: Inflammatory related cardiovascular disease, i.e. cardiac allograft rejection, myocarditis, septic shock, are accompanied by cytokine production, which stimulates the expression of inducible nitric oxide (iNOS). MATERIALS AND METHODS: The aim of the present study was to examine whether anti-inflammatory doses of acetylsalicylic acid (aspirin) could regulate iNOS protein expression in bovine vascular smooth muscle cells (BVSMCs) in culture. RESULTS: Interleukin 1 beta (IL-1 beta, 0.03 U mL-1) induced nitric oxide release by BVSMCs. Aspirin inhibited nitric oxide release from IL-1 beta-stimulated BVSMCs in a dose-dependent manner. In addition, aspirin significantly inhibited iNOS protein expression in BVSMCs and reduced the translocation of the nuclear factor-kappa B (NF-kappa B). Furthermore, aspirin and the blockade of NO generation by BVSMCs reduced the production of tumour necrosis factor alpha (TNF-alpha) by these cells. CONCLUSION: High doses of aspirin inhibited iNOS protein expression in BVSMCs and decreased NF-kappa B mobilization. The inhibition of iNOS expression by aspirin was further associated with a reduced ability of BVSMCs to produce TNF-alpha. This study could provide new mechanisms of action for aspirin in the treatment of the inflammation-related cardiovascular diseases.     

The role of nitric oxide synthase/nitric oxide in vascular smooth muscle control

Vascular compliance is dependent on endogenous and exogenous sources of nitric oxide (NO). In a discussion of therapeutics and NO derived via nitric oxide synthase (NOS) enzymes, it is necessary to examine the pathways of each drug to provide the clinical perfusionist with a greater understanding of the role of NOS/NO in vascular function. Endothelial-derived NO is a contributor in the vasoregulation of vascular smooth muscle. Therapeutics seek to mimic the vasodilatory effects of the endogenous NO. The therapeutics included in this review are nitroglycerin, nitroprusside, amyl nitrite, and inhalation of NO. L-Arginine supplementation provides additional substrate for the endogenous pathway that can augment NO production. NO is a small bioactive molecule involved in various biochemical pathways. Dysregulation of NO production can impair normal physiologic control of vascular compliance. Therefore, the purpose of this review is to provide the perfusionist with an understanding of the biochemical and pharmacological aspects of NOS/NO associated with vascular function.     

Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritides.

In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05). Immunohistochemical analysis as well as in situ hybridization showed that inducible NO synthase was predominantly expressed in synovial lining cells, endothelial cells, chondrocytes, and to a lesser extent, in infiltrating mononuclear cells and synovial fibroblasts. The synovial lining cells and the infiltrating cells expressing inducible NO synthase were identified where CD14+ cells were located. Together with morphological features, this suggests that they are type A synoviocytes. NO production from freshly isolated synoviocytes and chondrocytes was up-regulated by in vitro stimulation with a combination of IL-TNF-beta, TNF-alpha, and LPS. In summary, the present results suggest that NO is produced primarily by CD14+ synoviocytes, chondrocytes, and endothelial cells in inflammatory joints of arthritides. NO production can be upregulated by cytokines present in inflamed joints. The increased NO production may thus contribute to the pathological features in inflammatory arthritides.     

Induction of nitric oxide synthase by cyclic AMP in rat vascular smooth muscle cells.

By measurements of NO2-/NO3- (NOx) production and Northern blot analysis, we studied the effects of a membrane-permeable cAMP derivative, 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) gene and the synthesis of NOx in cultured rat vascular smooth muscle cells (VSMCs). 8-bromo-cAMP stimulated NOx production and increased steady-state levels of iNOS mRNA in rat VSMC in a time- and dose-dependent manner. NG-monomethyl-L-arginine, a NOS inhibitor, completely blocked the 8-bromo-cAMP-induced NOx production, whose effect was partially, but significantly reversed by an excess L-arginine, but not by D-arginine. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine), all stimulated NOx production. Dexamethasone inhibited the stimulated NOx production, as well as the induction of iNOS mRNA by cAMP. Both actinomycin D and cycloheximide completely blocked the stimulated NOx production by cAMP. Actinomycin D abolished the cAMP-induced iNOS mRNA, whereas cycloheximide remarkably increased iNOS mRNA levels in the presence and absence of 8-bromo-cAMP (superinduction). Actinomycin D, but not dexamethasone, completely abolished the cycloheximide-induced iNOS mRNA. The half-life of cAMP-induced iNOS mRNA was approximately 2 h, whereas no decay in the cycloheximide-induced iNOS mRNA was observed during 12 h. These results demonstrate that iNOS gene is upregulated by cAMP and the superinduction of iNOS mRNA is attributable to increased mRNA stability in rat VSMC.     

八肽缩胆囊素对脂多糖诱导血管内皮细胞诱生型一氧化氮合酶表达变化的抑制作用

目的探讨八肽缩胆囊素（CCK-8）对脂多糖（LPS）诱导血管内皮细胞诱生型一氧化氯合酶（iNOS）表达变化的影响。方法培养人脐静脉内皮细胞株ECV-304细胞。用0．01、0．1和1mg／L LPS处理2～24h，用生理盐水、10mol／LCCK-8和0．1mg／L LPS＋10^-8、10^-7、10^-8mol／L CCK-8处理16h；用比色法检测培养液中一氧化氮（NO）含量、细胞NOS活性，免疫细胞化学及蛋白质免疫印迹法检测iNOS蛋白表达。结果与生理盐水处理的对照组比较，LPS诱导培养液NO含量增多、细胞NOS活性增高、iNOS蛋白表达上调；CCK-8剂量依赣性抑制LPS的上述效应。而单独作用对iNOS蛋白表达、NOS活性和NO含量均无明显影响。结论CCK-8可以明显抑制LPS引起ECV-304细胞iNOS蛋白表达上调。减少NO生成。     

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.     

Evidence for inducible nitric-oxide synthase expression and activity in vascular smooth muscle of streptozotocin-diabetic rats

Experiments were performed to investigate whether nitric-oxide synthase (NOS) activity can be detected in vascular smooth muscle (VSM) from 12- to 14-week streptozotocin (STZ)-diabetic rats. Concentration-response curves to norepinephrine (NE) of superior mesenteric arteries from diabetic and age- and gender-matched control rats were obtained in the presence of dexamethasone (0.1 microM) to prevent in vitro induction of iNOS. Incubation of endothelium-intact arteries from diabetic rats with the nonselective NOS inhibitor, N(5)-(1-iminoethyl)L-ornithine (L-NIO) (300 microM), increased the NE sensitivity (expressed as the pD2 or -log EC50) from 6.58 +/- 0.05 to 8.39 +/- 0.12 (mean +/- S.E.M., n = 8). L-NIO produced a significantly smaller increase in the NE pD2 value in endothelium-intact arteries from control rats (from 6.51 +/- 0.03 to 7.08 +/- 0.03, p < 0.05). On endothelium removal, L-NIO still increased the NE pD2 value in diabetic arteries, from 7.48 +/- 0.03 to 8.38 +/- 0.15 (p < 0.05), but had no effect in control arteries. The selective iNOS inhibitor S-ethylisothiourea (EIT), but not the selective nNOS inhibitor 7-nitroindazole (7-NINA), produced an increase in the NE pD2 value in endothelium-denuded mesenteric arteries from diabetic but not control rats. Immunohistochemical staining indicated the presence of iNOS (but not eNOS or nNOS) in the medial and adventitial layers of mesenteric arteries from diabetic but not control rats. Quantitative measurement of cytosolic NOS activity indicated no significant calcium-dependent (nNOS) activity in control or diabetic arteries, or calcium-independent (iNOS) activity in control arteries. However, significant calcium-independent (iNOS) activity was detected in diabetic arteries. These data suggest that iNOS is functionally expressed in VSM of arteries from 12- to 14-week STZ-diabetic rats. The possible causes and consequences of the iNOS induction are discussed.     

Sphingosine 1-phosphate inhibits nitric oxide production induced by interleukin-1beta in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) is a lipid mediator that exerts potent and diverse biological effects on several cardiovascular cells. We investigated the effect of S1P on interleukin (IL)-1beta-induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in rat vascular smooth muscle cells (VSMCs). S1P inhibited NO production at concentrations higher than 0.1 muM; this was associated with the inhibition of iNOS protein and mRNA expression. S1P also inhibited IL-1beta-induced GTP cyclohydrolase I (GTPCH) mRNA expression. Pertussis toxin (PTX) partially attenuated the inhibitory effects of S1P on NO production and iNOS protein induction, whereas it completely blocked the inhibitory effects on iNOS and GTPCH mRNA expression. S1P inhibited iNOS expression in Ca(2+)-depleted conditions; PTX did not modify this effect. The Rho kinase inhibitor Y 27632 partially but significantly attenuated the inhibitory effect of S1P on iNOS expression in Ca(2+)-depleted condition but did not affect it in the presence of Ca(2+). S1P significantly inhibited IL-1beta-induced persistent activation of extracellular signal-regulated kinase (ERK) but had no effect in Ca(2+)-depleted conditions. Thus, S1P inhibits IL-1beta induction of NO production and iNOS expression in rat VSMCs through multiple mechanisms involving both PTX-sensitive and -insensitive G proteins coupled to S1P receptors. Furthermore, Ca(2+)-dependent ERK inhibition and Ca(2+)-independent Rho kinase activation might be involved in the inhibitory mechanism of iNOS expression. Through its action on NO production by VSMCs, S1P may play an important role in the progression of local vascular injury associated with thrombosis, atherosclerosis, and hypertension.     

降钙素基因相关肽对高血压病患者血管平滑肌细胞一氧化氮合酶的影响

目的探讨降钙素基因相关肽(CGRP)对高血压病(EH)患者动脉血管平滑肌细胞(VSMC)的一氧化氮合酶(NOS)、一氧化氮(NO)产生和细胞增殖的作用.方法对高血压病患者和血压正常者(NT)的动脉VSMC进行培养,分别测定两组VSMC的NO含量、NOS活性、细胞数量和细胞周期,观察高血压病患者VSMC的NO产生和细胞增殖以及CGRP对其的影响.结果①高血压病患者VSMC的NO含量和NOS活性分别为(42.73±6.76)μmol/2.5×106 cells和(0.24±0.05)nmol/min,均显著低于血压正常对照组的(74.52±4.37)μmol/2.5×106 cells和(0.55±0.10)nmol/min,P均＜0.01;细胞数量、细胞周期中的S期百分率和增殖指数(PI)分别为(10.39±1.30)×106个/ml,(35.78±1.0)%和(54.84±1.98)%,均显著高于血压正常对照组的(7.67±0.98)×106个/ml,(28.88±1.07)%和(44.12±1.43)%(P均＜0.01).②在CGRP干预下,高血压病患者VSMC的NO含量和NOS活性均较血压正常对照组为低(P＜0.01);细胞数量、细胞周期中的S期百分率和PI均较血压正常对照组为高(P均＜0.01).③在CGRP干预下,两组VSMC的NO含量和NOS活性均较基础状态显著增加和增强(P＜0.01);两组VSMC的细胞数量、细胞周期中的S期百分率和PI均较基础状态显著减低(P＜0.01).结论高血压病患者VSMC的NOS-NO系统可能存在功能或结构的异常,并对CGRP的反应性降低,这可能是高血压的发病原因之一.     

活血化瘀汤对骨折愈合过程中诱导型一氧化氮合酶基因表达的影响

目的:观察活血化瘀汤对骨折愈合中诱导型一氧化氮合酶mRNA表达的影响,从而调控成骨作用。方法:实验于2002-12/2004-08在华中科技大学基础学院分子生物学实验室完成。将24只SD大鼠胫骨中段闭合骨折骨髓内克氏针固定制成骨折模型,随机分为活血化瘀汤组(n=12)及生理盐水组(n=12);于骨折后4,7,14,21d收集骨痂样本,反转录聚合酶链反应测定诱导型一氧化氮合酶的mRNA表达水平。结果:24只大鼠均进入结果分析。骨折后7,14,21d所有骨痂样本诱导型一氧化氮合酶的mRNA表达增强,骨折后14d达到峰值,活血化瘀汤组与生理盐水组比较,骨折后7,14d骨痂诱导型一氧化氮合酶基因表达显著性增强(P=0.0037)。结论:活血化瘀汤可能通过增加早期大鼠骨痂诱导型一氧化氮合酶源性一氧化氮,影响骨细胞增殖功能,从而促进骨折愈合。     

Specific effect of arachidonic acid on inducible nitric oxide synthase mRNA expression in human osteoblastic cells

A specific modulatory effect of PUFAs (polyunsaturated fatty acids) on gene expression of some cytokines involved in bone remodelling has been reported previously. In particular, although a direct action of AA (arachidonic acid) on bone cytokine gene expression has been shown in human osteoblastic cells, OA (oleic acid) and EPA (eicosapentaenoic acid) were ineffective. Since the NO (nitric oxide) system has also been shown to have an important modulatory activity on osteoblasts, osteoclasts and bone metabolism, in the present study we have investigated the effects of PUFAs on iNOS (inducible NO synthase) gene expression in a human osteoblast-like cell line. AA induced a significant increase in iNOS mRNA expression, whereas EPA and OA had no stimulatory effects but instead caused a significant inhibition of AA-induced iNOS gene expression. Blocking of the COX (cyclo-oxygenase) pathway did not inhibit AA-induced iNOS expression. AA action was inhibited instead by the addition of calphostin C and genistein, inhibitors of PKC (protein kinase C) and tyrosine kinases respectively. Experiments performed with specific anti-cytokine antibodies showed a significant decrease in iNOS expression in AA-treated osteoblastic cells, suggesting that both cytokine-dependent and -independent mechanisms account for the effects of AA on iNOS gene expression. In conclusion, our investigation clearly shows specific effects of PUFAs on iNOS expression in human osteoblast-like cells with a cytokine-dependent and -independent mechanism. These results might have clinical relevance and are of interest for understanding the reported beneficial effects of dietary PUFA manipulation on the prevention and/or treatment of primary and secondary bone disease.     

Circulating factor in patients with recurrent focal segmental glomerulosclerosis postrenal transplantation inhibits expression of inducible nitric oxide synthase and nitric oxide production by cultured rat mesangial cells.

BACKGROUND: Primary focal segmental glomerulosclerosis (FSGS) recurs in nearly 30% of patients who progress to end-stage renal disease and then receive a kidney transplant. A circulating plasma factor has been isolated from these patients that increases glomerular permeability to albumin in vitro. Because of the pivotal role of the mesangial cell in the accumulation of extracellular matrix (ECM) material within the glomerulus and the modulation of matrix protein synthesis by nitric oxide (NO), we examined the effect of the FSGS factor on inducible nitric oxide synthase (iNOS) expression and NO production by cultured rat mesangial cells (RMC). METHODS: RMC were incubated with the supernatant following 70% ammonium sulfate precipitation of serum from patients with recurrent FSGS. RESULTS: Addition of the FSGS factor to cultured RMC led to a significant inhibition of nitrite accumulation, an index of NO synthesis. There was a parallel decline in iNOS gene and protein expression. Sera obtained from control patients or those with minimal change nephrotic syndrome or diabetic nephropathy that was processed in the same manner as FSGS samples had no effect NO synthesis or iNOS activity. The inhibitory effect of the FSGS factor on NO production persisted despite addition of indomethacin (0.1-1 mumol/L) or cyclosporine (25 micrograms/mL) to test media. CONCLUSIONS: These data indicate that the FSGS factor independently alters two aspects of glomerular function--permselectivity and matrix protein synthesis--by distinct mechanisms. FSGS factor-induced disturbances in iNOS gene and protein expression and NO production by mesangial cells may antagonize the antifibrotic effect of NO within the mesangium and contribute to progressive glomerulosclerosis in patients with primary FSGS.     

Inducible interleukin-1 gene expression in human vascular smooth muscle cells.

Interleukin-1 (IL-1) mediates many components of generalized host response to injury and may also contribute to local vascular pathology during immune or inflammatory responses. Because altered function of smooth muscle cells (SMC) accompanies certain vascular diseases, we tested whether SMC themselves might produce this hormone. Unstimulated SMC contain little or no IL-1 mRNA. However, exposure to bacterial endotoxin caused accumulation of IL-1 mRNA in SMC cultured from human vessels. Endotoxin maximally increased IL-1 beta mRNA in SMC after 4-6 h. The lowest effective concentration of endotoxin was 10 pg/ml. 10 ng/ml produced maximal increases in IL-1 beta mRNA. Interleukin-1 alpha mRNA was detected when SMC were incubated with endotoxin under "superinduction" conditions with cycloheximide. Endotoxin-stimulated SMC also released biologically functional IL-1, measured as thymocyte costimulation activity inhibitable by anti-IL-1 antibody. Thus, human SMC can express IL-1 beta and IL-1 alpha genes, or very similar ones, and secrete biologically active product in response to a pathological stimulus. Endogenous local production of this inflammatory mediator by the blood vessel wall's major cell type could play an important early role in the pathogenesis of vasculitis and arteriosclerosis.     

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。     

肾血管性高血压对诱导型一氧化氮合酶表达及活性的影响

目的通过测定肾血管性高血压大鼠血管及肾组织诱导型一氧化氮合酶（iNOS）活性及表达的变化情况,探讨血压与iNOS间的关系。方法运用肾动脉不全结扎方法制备SD大鼠肾血管性高血压模型,并应用Greiss反应、L-精氨酸同位素标记法及Westernblot等分别测定一氧化氮的终产物——尿中NO     

Thermal injury-induced peroxynitrite production and pulmonary inducible nitric oxide synthase expression depend on JNK/AP-1 signaling

OBJECTIVE: To determine whether burn-induced peroxynitrite production and expression of lung inducible nitric oxide synthase (iNOS), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, CXCR2, macrophage inflammatory protein (MIP)-2, and neutrophil chemokine (KC) are mediated by the c-Jun NH2-terminal kinase (JNK). DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the mice. INTERVENTIONS: In experiment 1, specific pathogen-free C57/BL6 mice were subjected to 30% total body surface area third-degree burn over shaved back. At 0 hr, 2 hrs, 4 hrs, and 6 hrs after burn, lung tissues of those mice were harvested for JNK activity assay, AP-1 DNA-binding activity, and pJNK immunohistochemistry. In experiment 2, a specific JNK inhibitor, SP600125, was given (30 mg/kg intraperitoneally) to mice immediately postburn to suppress the JNK activity. At 8 hrs after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine (DHR) 123 oxidation. Lung tissues were harvested for myeloperoxidase (MPO) determination, ICAM-1, VCAM-1, CXCR2, KC, MIP-2, interleukin-1beta, and interleukin-6 messenger RNA expression; iNOS immunohistochemical staining; and histologic studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. MEASUREMENTS AND MAIN RESULTS: The JNK activity and AP-1 DNA-binding activity of lung tissue significantly increased to a peak at 2 hrs and 4 hrs, respectively, after thermal injury. Immunohistochemical study demonstrated that the increase of the pJNK was mostly from the bronchiole epithelial cells. This increase of MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased six-fold, nine-fold, and four-fold after burn. SP600125 administration obliterated the thermal injury-induced JNK activity, AP-1 DNA-binding activity, and iNOS expression in lung tissue. SP600125 treatment also significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 54%, 8%, and 47%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. Furthermore, SP600125 abolished thermal injury-induced ICAM-1, VCAM-1, CXCR2, MIP-2, and KC but not interleukin-1beta and interleukin-6 messenger RNA levels of lung tissues. CONCLUSIONS: Thermal injury induces lung tissue JNK activation and AP-1 DNA-binding activity mainly from airway epithelial cells. Thermal injury-induced peroxynitrite production and lung iNOS, ICAM-1, and VCAM-1 expression are mediated by the JNK signaling. JNK inhibition decreases thermal injury-induced lung neutrophil infiltration and subsequently pulmonary hyperpermeability.     

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.     

1-alpha,25-Dihydroxyvitamin D3 regulates inducible nitric oxide synthase messenger RNA expression and nitric oxide release in macrophage-like RAW 264.7 cells

The expression of inducible nitric oxide synthase (iNOS) expression and release of nitric oxide (NO) from macrophages are markedly increased in granulomatous infections. Activation of macrophages 1alpha-hydroxylase results in an increase of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, the significance of this increased production is not completely understood. In this study, we analyzed 1,25(OH)(2)D(3) and NO production in patients with tuberculosis infection and hypercalcemia and used lipopolysaccharide (LPS) to stimulate RAW 264.7 cells in an attempt to assess iNOS expression and gaseous NO production regulated by 1,25(OH)(2)D(3). Peroxynitrite (OONO(-)) production and lactate dehydrogenase activity were also examined. Without additional stimulation, peripheral-blood mononuclear cells (PBMCs) from patients with tuberculosis converted more 25-hydroxyvitamin D(3) to 1,25(OH)(2)D(3) than did those from normal controls. These PBMCs released less NO than did those from control subjects, at baseline and in the stimulated state. We found that 1,25(OH)(2)D(3) dose-dependently inhibited iNOS messenger RNA expression of the LPS-stimulated RAW 264.7 cells and also significantly reduced the gaseous NO release and OONO(-) production. Paralleling the 1,25(OH)(2)D(3)-induced inhibition of NO release were reductions in OONO(-) and LDH production. In conclusion, 1,25(OH)(2)D(3) inhibited iNOS expression and reduced NO production by LPS-stimulated macrophages in the range of physiological doses. Inhibition of the NO surge was coupled with a reduction in OONO(-) and LDH production. Increased 1,25(OH)(2)D(3) production and decreased release of NO from the PBMCs of patients with tuberculosis and hypercalcemia were also noted. We propose that 1,25(OH)(2)D(3) production by macrophages may protect themselves against oxidative injuries caused by the NO burst. In the case of tuberculosis infection, increased 1,25(OH)(2)D(3) synthesis may further contribute to the development of an unwanted phenomenon-hypercalcemia.