Endothelial nitric oxide synthase gene polymorphisms in Fabry's disease

The gene encoding endothelial nitric oxide synthase (eNOS) is involved in abnormalities in nitric oxide (NO) synthesis that mediates functional damage of vascular cells, especially of endothelial cells (ECs), a common characteristic in cardiovascular diseases. In Fabry's disease, the characteristic mutation in the alpha-galactosidase A (alpha-gal A) gene induces large deposits of glycosphingolipids, particularly concentrated in ECs, a process associated with endothelial dysfunction. To determine whether in addition to alpha-gal A gene mutations, eNOS genetic variations are implicated in this process, we examined the genotypes of the missense Glu298Asp (G894T) variant in exon 7 and 27-bp tandem repeats in intron 4 (4b/a) in 19 patients with Fabry's disease, and 39 normal volunteers. The results showed that both varials have a significant association with Fabry's disease. The frequencies of mutant Glu/Asp + Asp/Asp genotypes and Asp allele are significantly higher in Fabry's disease (68.4%, p = 0.044, and 47.4%, p = 0.022, respectively) than in controls (46.7% and 25%, respectively). The frequencies of eNOS 4b/a polymorphisms are also significantly different in Fabry's disease when compared to controls. The mutant 4b/a + 4a/a genotype frequencies are 55.5% (p = 0.032) and 4a allele 27.8% (p = 0.05) compared with controls (23.1% and 12.8%, respectively). These results indicate that more than half of the patients with Fabry's disease carry the Glu298Asp variant ( approximately 68%) and/or the 4b/a polymorphism ( approximately 55%). To the best of our knowledge, this is the first report showing an influence of eNOS gene polymorphisms in patients with Fabry's disease.     

Endothelial nitric oxide synthase gene polymorphisms and prostate cancer risk in Serbian population

Genome-wide association studies (GWAS) have identified over 46 SNPs associated with human prostate cancer (PCa). Some studies have shown correlation of the nitric oxide synthase (NOS) NOS3 gene polymorphisms with the risk and/or progression of PCa. This study aimed to evaluate the association of NOS3 gene polymorphisms (−786T>C, −764A>G, −714G>T, −690C>T, −649G>A and 894G>T) with PCa risk and progression. 150 patients with PCa, 150 patients with BPH and 100 age-matched healthy controls were recruited in this study. Genotyping of promoter polymorphisms was performed by bi-directional DNA sequencing, and for 894G>T by RFLP analysis. There was no significant association between the alleles and genotypes of these genetic variants and PCa risk. For −786T>C polymorphism, we found that C allele is associated with absence of metastases, assuming dominant genetic model (P = 0.049; OR, 0.50; 95% CI, 0.25–1.00). It was found that, compared with NOS3 −690C>T variant CC genotype, CT and TT genotypes confer decreased risk of developing metastases (dominant model, P = 0.015, OR, 0.24; 95% CI, 0.07–0.88) and show association with low clinical tumour stage, compared with stages T₃ and T₄ (dominant model, P = 0.046, OR, 0.20; 95% CI, 0.04–1.02). Genetic variants −764A>G, −714G>T, −649G>A were not detected in our study group. There is evidence of an inverse correlation of the NOS3 894G>T minor allele with high serum PSA (>20 ng/ml) (dominant model, P = 0.013, OR, 0.37; 95% CI, 0.17–0.82). Our results suggest that NOS3 gene polymorphisms are genetic susceptibility factors for the progression of PCa and patient outcome.     

Endothelial nitric oxide synthase gene sequence variations and vascular disease

Genes contribute significantly to interpopulation differences in vascular disease. Endothelial nitric oxide synthase (eNOS)-a key regulator of vascular nitric oxide production-has been investigated extensively to determine the relevance of DNA variants in the eNOS gene and vascular diseases. Variants in the promoter region, introns, and exons have been explored in a large number of populations but findings have been inconsistent. This paper reviews the current status of functional significance for reported sequence variations in the eNOS gene and the relevance of these variants to different forms of vascular diseases.     

Endothelial nitric oxide synthase gene polymorphism in women with idiopathic recurrent miscarriage

BACKGROUND: Lack of endothelium-derived nitric oxide is associated with vasospasm and vascular infarction. We investigated the relationship between idiopathic recurrent miscarriage and a polymorphism of the gene encoding endothelial nitric oxide synthase (NOS3). METHOD: In a prospective case-control study, 105 women with idiopathic recurrent miscarriage and 91 healthy controls were investigated. We used the polymerase chain reaction to identify the different alleles of a 27 base pair tandem repeat polymorphism in intron 4 of the NOS3 gene. RESULTS: The wild type B allele was identified on 329 out of 392 chromosomes (frequency 0.84). The polymorphic A allele was present on 63 chromosomes (frequency 0.16). The genotype frequencies were as follows: 68% (B/B), 31% (A/B) and.5% (A/A). The distribution of genotype frequencies was significantly different between the study and control groups for allele A/B heterozygotes (NOS3(A/B)) (36.7 versus 23.8%, P = 0.03, OR 1.6, 95% CI 1.1--3.8). Only one individual was homozygous for the A allele (NOS3(A/A)). She was in the study group. Between women with primary and secondary recurrent miscarriages, no statistically significant difference between the distribution of NOS3(A/B) genotypes (28 versus 34%) was observed. CONCLUSIONS: These data support a role for the NOS3 gene as a genetic determinant of the risk of idiopathic recurrent miscarriage.     

Endothelial nitric oxide gene polymorphisms,nitric oxide production and coronary artery disease risk in a South Indian population

Nitric oxide (NO) synthesized by vascular endothelial cells, is a vasodilator agent produced from endothelial NO synthase (eNOS). It has been reported that decreased bioavailability of NO plays an important role in the development and progression of atherosclerosis.Electrocardiographically proven 100 patients with acute myocardial infarction and 100 age and sex matched healthy individuals with normal coronary arteries were included for the study. The genotypes of a 27-bp insertion/deletion in intron 4 (eNOS 4b/4a) and G894T polymorphism in exon 7, were determined by PCR analysis based on the banding pattern on gel electrophoresis. The genotype frequencies were calculated following the Hardy–Weinberg law. Serum NO level was also estimated by the Griess method. NO levels in AMI patients were higher than those of the healthy subjects (median [interquartile range], (14.36[12.42–15.78]) μM compared with 11.28[10.32–11.89]) μM; p < 0.001; Mann–Whitney rank sum test, U = 285. Mutant “T” allele frequency of the eNOS-G894T polymorphism was found to be comparatively higher (0.29) in AMI patients than among the controls (0.17). The calculated Odds ratio showed that the occurrence of mutant allele “T” was 1.6 fold as frequent in cases than controls [OR = 1.6 (95%CI 0.898 to 2.833)].To conclude, in the present study, (i) NO levels were found to be increased in patients than in controls, (ii) the homozygous mutant (TT) genotype confers genetic susceptibility to coronary artery disease (iii) both the eNOS 4a/b and G894T polymorphisms were not associated with serum NO levels in a South Indian Tamil population.     

Endothelial nitric oxide synthase gene (NOS3) variant and hypertension in pregnancy

Hypertension in pregnancy (HP), including preeclampsia, is known to be a multifactorial disease. Recently, a Glu298Asp variant of the endothelial nitric oxide synthase gene (NOS3) was identified as being associated with coronary spasm and myocardial infarction, whereas it has been reported that endothelial nitric oxide synthase plays a role in HP. We therefore performed an association study of the Glu298Asp variant with HP among 152 HP patients and 335 normal pregnant control individuals, in the context of other risk factors before pregnancy. The frequency of the variant GA+AA NOS3 genotypes was significantly higher in the patients (0.23) than in the controls (0.12) (P < 0.01). Multivariate analysis revealed that family history of hypertension, TT genotype of the angiotensinogen gene (AGT), GA+AA NOS3 genotype, and prepregnancy body mass index ≥ 24 were independent potent risk factors, after adjustment for maternal age and parity. The odds ratios of the factors were 2.7, 2.3, 2.2, and 2.1, respectively. Our results suggested that the Asp298 of NOS3 is a potent, independent risk factor for HP. © 2001 Wiley‐Liss, Inc.     

Endothelial nitric oxide synthase (eNOS) expression and localization in healthy and diabetic rat hearts

Several studies suggest that nitric oxide (NO) production is reduced in diabetes and that the decrease of NO may be related to the pathogenesis of diabetic endothelial damage. NO synthase (NOS) catalyses the conversion of L-arginine to L-citrulline in the presence of oxygen and NADPH-diaphorase (NADPH-d). In this study, we evaluated the expression of endothelial NOS (eNOS) enzyme and its co-enzyme in diabetic rat hearts. Male Wistar rats (n = 20, 4 mo old) and 20 male Bio Breeding Wistar (BB/W) rats of the same age were used; the Wistar rats represent the control non-diabetic rats while the BB/W rats represent the diabetic group. After the hearts were excised, the NADPH-d co-enzyme was visualized by a histochemical method and the endothelial isoform of NOS was localized by immunohistochemistry. In addition, eNOS gene expression was estimated by rt-PCR, and eNOS protein level was detected by Western blot analysis. The eNOS visualization, which involved immunoprecipitation, and the NADPH-d visualization, which involved histochemical staining, were both diminished in endothelial cells of the vascular wall of diabetic hearts, compared to non-diabetic hearts. The eNOS protein level, evaluated by Western blotting, was evident as an intense band in cardiac homogenates of non-diabetic and diabetic rats. The expression of mRNA for eNOS did not differ significantly between the two groups. These findings indicate that, in this rat heart model, diabetes does not influence the overall eNOS protein level or its mRNA level. However, there a diminution in the deposition of eNOS in cardiac endothelial cells of diabetic rats, versus non-diabetic controls, suggesting a relation between eNOS and the loss of vasodilatory response that is observed in diabetes.     

Prevalence of endothelial nitric oxide synthase gene polymorphisms in patients with atopic asthma

BACKGROUND: Asthma is a common multifactorial disease, the aetiology of which is attributable to both environmental and genetic factors. The endothelial nitric oxide synthase (NOS3) gene has been implicated in asthma pathogenesis. OBJECTIVE: This study investigated associations of 27 base-pair tandem repeat polymorphism in intron 4 and the Glu298Asp (G894T) variant of the NOS3 gene with atopic asthma in a Czech population. METHODS: Polymerase chain reaction was used to determine the NOS3 genotypes in subjects with atopic asthma (n = 163) and random controls (n = 209). RESULTS: The NOS3 allele or genotype distributions did not differ significantly between the control and asthma groups. However, the common genotype (bb) of the NOS3 polymorphism in intron 4 was found to be significantly associated with total IgE levels (P = 0.006), specific IgE levels for feathers (P = 0.0002) and a positive skin prick test for hay (P = 0.004). In one atopic patient, we identified an additional 27-bp repeat in the NOS3 gene (NOS3c), which occurred in heterozygous combination with the NOS3b allele (NOS3b/c genotype). In addition, we describe a new polymorphism (A5495G) in the NOS3 gene, which was in almost complete linkage disequilibrium with the NOS3 repeat polymorphism in intron 4. The Glu298Asp variant was not associated with asthma and/or related atopic phenotypes in our study. CONCLUSION: Neither the NOS3 'b' allele nor the NOS3 'b/b' genotype showed any general association with atopic asthma, but they were associated with atopy-related phenotypes. We conclude that the NOS3 gene polymorphisms may act as disease modifiers in atopic asthma phenotype in our population.     

Endothelial nitric oxide synthase expression in pulmonary capillary hemangiomatosis

Pulmonary capillary hemangiomatosis (PCH) is an unusual disorder characterized by the proliferation of capillaries in the alveolar septa and pulmonary interstitium. Originally conceived as a primary idiopathic disorder of the pulmonary microcirculation, recent studies have demonstrated that PCH may be associated with other pathologies. Nitric oxide (NO) is a gaseous free radical with protean biological effects that is released during the intracellular conversion of arginine to citrulline. Nitric oxide synthases (NOS) mediate the production of NO and the release of NO in the microvasculature is specifically catalyzed by endothelial NOS (NOS-III). As NOS contributes to angiogenesis and is reduced in the hypertensive pulmonary microcirculation, we examined the expression of NOS-III protein in situ in the lungs of patients with PCH. Reduced microvascular expression of NOS-III protein by endothelial cells was observed in 4/6 (67%) cases of PCH, and all of these showed concomitant pulmonary vascular hypertensive remodeling. In 2/6 (33%) cases of PCH with no morphologic evidence of pulmonary hypertensive arteriopathy, endothelial expression of NOS-III protein was judged to be either minimally reduced or normal. These findings suggest that NOS-III is specifically reduced in PCH when pulmonary arterial hypertensive remodeling is concomitantly present.     

Endothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo

Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin-1 scaffolding domain (AP-Cav) reduces eNOS-regulated hyperpermeability. We used eNOS-deficient (eNOS−/−) mice and their wild-type control as experimental animals, platelet-activating factor (PAF) at 10⁻⁷ m as the test pro-inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild-type cremaster muscle from a baseline of 2.4 ± 2.2 to a peak net value of 84.4 ± 2.7 units, while the corresponding values in cremaster muscle of eNOS−/− mice were 1.0 ± 0.3 and 15.6 ± 7.7 units ( P < 0.05). Similarly, PAF increased IOI in the mesentery of wild-type mice but much less in the mesentery of eNOS−/− mice. PAF increased IOI to comparable values in the mesenteries of wild-type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP-Cav blocked the microvascular hyperpermeability responses to 10⁻⁷ m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin-1 inhibits eNOS regulation of microvascular permeability in vivo . Our results establish eNOS as an important regulator of microvascular permeability in inflammation.     

Endothelial nitric oxide synthase transgenic models of endothelial dysfunction

Endothelial production of nitric oxide is critical to the regulation of vascular responses, including vascular tone and regional blood flow, leukocyte–endothelial interactions, platelet adhesion and aggregation, and vascular smooth muscle cell proliferation. A relative deficiency in the amount of bioavailable vascular NO results in endothelial dysfunction, with conditions that are conducive to the development of atherosclerosis: thrombosis, inflammation, neointimal proliferation, and vasoconstriction. This review focuses on mouse models of endothelial dysfunction caused by direct genetic modification of the endothelial nitric oxide synthase (eNOS) gene. We first describe the cardiovascular phenotypes of eNOS knockout mice, which are a model of total eNOS gene deficiency and thus the ultimate model of endothelial dysfunction. We then describe S1177A and S1177D eNOS mutant mice as mouse models with altered eNOS phosphorylation and therefore varying degrees of endothelial dysfunction. These include transgenic mice that carry the eNOS S1177A and S1177D transgenes, as well as knockin mice in which the endogenous eNOS gene has been mutated to carry the S1177A and S1177D mutations. Together, eNOS knockout mice and eNOS S1177 mutant mice are useful tools to study the effects of total genetic deficiency of eNOS as well as varying degrees of endothelial dysfunction caused by eNOS S1177 phosphorylation.     

Endothelial nitric oxide synthase gene (NOS3) variant and hypertension in pregnancy.

Hypertension in pregnancy (HP), including preeclampsia, is known to be a multifactorial disease. Recently, a Glu298Asp variant of the endothelial nitric oxide synthase gene (NOS3) was identified as being associated with coronary spasm and myocardial infarction, whereas it has been reported that endothelial nitric oxide synthase plays a role in HP. We therefore performed an association study of the Glu298Asp variant with HP among 152 HP patients and 335 normal pregnant control individuals, in the context of other risk factors before pregnancy. The frequency of the variant GA+AA NOS3 genotypes was significantly higher in the patients (0.23) than in the controls (0.12) (P < 0.01). Multivariate analysis revealed that family history of hypertension, TT genotype of the angiotensinogen gene (AGT), GA+AA NOS3 genotype, and prepregnancy body mass index > or = 24 were independent potent risk factors, after adjustment for maternal age and parity. The odds ratios of the factors were 2.7, 2.3, 2.2, and 2.1, respectively. Our results suggested that the Asp298 of NOS3 is a potent, independent risk factor for HP.     

Endothelial nitric oxide synthase (eNOS) genetic polymorphisms among Egyptian systemic lupus erythematosus and rheumatoid arthritis patients

Several genetic polymorphisms in the endothelial nitric oxide (eNOS) gene have been associated with different vascular, infectious, and autoimmune disease in some populations. These polymorphisms may alter the levels of eNOS expression, thus being relevant either to the pathogenesis or progression of these diseases. We aimed to assess the prevalence of eNOS genetic polymorphisms namely, the 27-bp repeat in intron 4 by polymerase chain reaction (PCR) and T-786C mutation by PCR-RFLP, among Egyptian systemic lupus erythematosus patients (SLE) and rheumatoid arthritis (RA) patients. The study included 34 SLE and 34 RA patients. Forty-six age, sex, and ethnically matched healthy volunteers were included in the current study as a control group. The frequencies of intron 4 and T-786C polymorphisms in the SLE patient group were 73.5 % for the wild type versus 26.5 % for the polymorphic types and 38.2 % for the wild type versus 50 % for the heteromutant type and 11.8 % for the homomutant type, respectively. As for the RA group the frequencies of intron 4 and T-786C polymorphisms in the patient group were 76.5 % for the wild type versus 23.5 % for the polymorphic types and 44.1 % for the wild type versus 55.9 % for the polymorphic type, respectively, with no statistically significant difference in the distribution of the genotypes between SLE and RA patients and the control group. No association was detected between these genetic polymorphisms and the clinical features of the patients apart from an association between the T-786C genetic polymorphism and the extra-articular manifestations of RA.     

Endothelial nitric oxide synthase, caveolae and the development of atherosclerosis

Early hypercholesterolaemia-induced vascular disease is characterized by an attenuated capacity for endothelial production of the antiatherogenic molecule nitric oxide (NO), which is generated by endothelial NO synthase (eNOS). In recent studies we have determined the impact of lipoproteins on eNOS subcellular localization and action, thereby providing a causal link between cholesterol status and initial abnormalities in endothelial function. We have demonstrated that eNOS is normally targeted to cholesterol-enriched caveolae where it resides in a signalling module. Oxidized low density lipoprotein (LDL; oxLDL) causes displacement of eNOS from caveolae by binding to endothelial cell CD36 receptors and by depleting caveolae cholesterol content, resulting in the disruption of eNOS activation. The adverse effects of oxLDL are fully prevented by high density lipoprotein (HDL) via binding to scavenger receptor BI (SR-BI), which is colocalized with eNOS in endothelial caveolae. This occurs through the maintenance of caveolae cholesterol content by cholesterol ester uptake from HDL. As importantly, HDL binding to SR-BI causes robust stimulation of eNOS activity in endothelial cells, and this process is further demonstrable in isolated endothelial cell caveolae. HDL also enhances endothelium- and NO-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, lipoproteins have potent effects on eNOS function in caveolae via actions on both membrane cholesterol homeostasis and the level of activation of the enzyme. These processes may be critically involved in the earliest phases of atherogenesis, which recent studies suggest may occur during fetal life.     

CD14 C-260T gene polymorphism and ischemic heart disease susceptibility: A HuGE review and meta-analysis

The CD14 gene C-260T polymorphism has been reported to be associated with ischemic heart disease, but results were conflicting. To evaluate the role of the CD14 C-260T polymorphism in ischemic heart disease, we performed meta-analyses of all available data. Comprehensive searches for studies on the association between the genotypes (CC, CT, TT) distributions and ischemic heart disease risk were performed. Patients with acute coronary syndrome, prior myocardial infarction, stable angina pectoris, or angiographic coronary artery stenosis were included. Potential sources of heterogeneity were explored by meta-regression. Analyses were performed under European, East Asian, and Indian studies, respectively. Data were available for 19 studies involving 11,813 cases and 6,196 controls. The summary odds ratio under the recessive model was 1.53 (95% confidence interval: 1.20–1.96) for East Asian studies published in English language journals on overall ischemic heart disease. Pooled odds ratios under the codominant model were about 1.81 (95% confidence interval: 1.36–2.40) and 1.70 (95% confidence interval: 1.26–2.29) for Chinese studies on overall ischemic heart disease and other ischemic heart disease (angina pectoris and angiographic coronary artery stenosis), respectively. No significant association was found in a European population, an Indian population, or the vulnerable plaque ischemic heart disease (acute coronary syndrome and prior myocardial infarction) subgroup of an East Asian population. It is probable that T allele and TT genotype are associated with ischemic heart disease in the East Asian population but not in the European or Indian populations. Further studies are warranted to assess these associations in greater details, especially in East Asian and Indian populations.     

Angiotensin II type 1 receptor polymorphisms and susceptibility to hypertension: A HuGE review

The angiotensin II type 1 receptor (AGTR1) plays an integral role in blood pressure control, and is implicated in the pathogenesis of hypertension. Polymorphisms within this gene have been extensively studied in association with hypertension; however, findings are conflicting. To clarify these data, we conducted a systematic review of association studies of AGTR1 polymorphisms and hypertension, and performed a meta-analysis of the rs5186 variant. Results show that the currently available literature is too heterogeneous to draw meaningful conclusions. The definition of hypertension and gender composition of individual studies helps to explain this heterogeneity. Although the structure and splicing pattern of AGTR1 would suggest a likely effect of polymorphisms within the promoter region on gene function, few studies have been conducted thus far. In conclusion, there is insufficient evidence that polymorphisms in the AGTR1 gene are risk factors for hypertension. However, most studies are inadequately powered, and larger well-designed studies of haplotypes are warranted.     

Analysis of the inducible nitric oxide synthase gene polymorphisms in Czech patients with atopic diseases

BACKGROUND: Nitric oxide (NO) is an important mediator of physiologic processes in the airways; it plays a significant role in the regulation of the T helper type 1/type 2 balance and contributes to the development of atopic diseases. OBJECTIVE: We analysed several polymorphisms mainly in the promoter region of the inducible NO synthase (NOS2, iNOS) gene and investigated their associations with asthma and/or atopic phenotypes. METHODS: We performed a case-control study in 994 subjects (661 patients with atopic disorders, with subgroups of 304 patients with allergic asthma, and 333 healthy individuals), matched for sex, living in the same geographical area. Screening for polymorphisms was performed by combination of PCR and direct sequencing analysis. RESULTS: We analysed 14 nucleotide sequence variants, seven most common of which were typed in quite large groups of our asthmatic, atopic and control populations. None of these seven frequent polymorphisms was associated with the phenotype bronchial asthma or other atopic diseases. Nevertheless, three from six common promoter polymorphisms showed a significant relation to feather's positivity (P value from 0.01 to 0.03) and the NOS2 608Leu variant was significantly associated with asthma severity [p(corr) = 0.0005; odds ratio (OR) = 5.00, 95% confidence interval (CI): 1.88-13.33]. In haplotype analysis, the most common -2447C/-1659C/-1026G/-0.7del/-277A/Ser608 haplotype was associated with a lower risk of asthma when compared with the common haplotypes with frequency more than 5% (P = 0.01, p(corr) < 0.05; OR = 0.65, 95% CI: 0.56-0.77). CONCLUSION: Our findings suggest that inducible NOS can play a role in atopic disorders, and several polymorphisms in its gene may be important for asthma protection or susceptibility.     

Cell-based gene transfer to the pulmonary vasculature: Endothelial nitric oxide synthase overexpression inhibits monocrotaline-induced pulmonary hypertension.

To circumvent the problems of in vivo transfection and avoid the use of viral vectors or proteins, we sought to establish whether smooth-muscle cells (SMCs) transfected ex vivo could be delivered via the systemic venous circulation into the pulmonary bed to achieve local transgene expression in the lung. Primary cultures of pulmonary artery SMCs from Fisher 344 rats were labeled with a fluorescent, membrane-impermeable dye chloromethyl trimethyl rhodamine or transfected with the beta-galactosidase (betaGal) reporter gene under the control of the cytomegalovirus (CMV) enhancer/promoter (pCMV-beta). Transfected or labeled SMCs (5 x 10(5) cells/animal) were delivered to syngeneic recipient rats by injection into the jugular vein; the animals were killed at intervals between 15 min and 2 wk; and the lungs, spleens, kidneys, and skeletal muscle were excised and examined. At 15 min after transplantation, injected cells were detected mainly in the lumen of small pulmonary arteries and arterioles, often in groups of three or more cells. After 24 h, labeled SMCs were found incorporated into the vascular wall of pulmonary arterioles, and transgene expression persisted in situ for 14 d with no evidence of immune response. Using simple geometric assumptions, it was calculated that approximately 57 +/- 5% of the labeled cells reintroduced into the venous circulation could be identified in the lungs after 15 min, 34 +/- 7% at 48 h, 16 +/- 3% at 1 wk, and 15 +/- 5% at 2 wk. Similar results were observed using cells transfected with the reporter gene betaGal. To determine whether this method of gene transfer could prove effective in inhibiting the development of pulmonary vascular disease, pulmonary artery SMCs were transfected with either the full-length coding sequence of endothelial nitric oxide synthase (NOS) under the control of the CMV enhancer/promoter or with the control vector (pcDNA3.1) and injected simultaneously with the pulmonary endothelial toxin monocrotaline. At 28 d after injection the right ventricular systolic pressure was significantly decreased from 50 +/- 4 mm Hg in animals injected with the null-transfected cells to 33 +/- 3 mm Hg in animals injected with the NOS-transfected cells (P < 0.01). These results suggest that a cell-based strategy of ex vivo transfection may provide an effective nonviral approach for the selective delivery of foreign transgenes to pulmonary microvessels in the treatment of pulmonary vascular disease.     

Endothelial nitric oxide synthase gene polymorphism in a cohort of Egyptian women with recurrent spontaneous miscarriage

The risk of miscarriage is enhanced by genetic and environmental factors. Several studies indicated that there was an association between endothelial nitric oxide synthase activity, implantation, and the maintenance of pregnancy. However, the results of these studies remain controversial. The aim of our study was to determine the association between recurrent spontaneous miscarriage (RSM) in Egyptian women and two common polymorphisms of the endothelial nitric oxide synthase (eNOS) gene: intron 4 and T-786C. The study was conducted on a total number of 100 participants, 50 patients with RSM and 50 age-matched healthy controls. Patients and controls were investigated for the two polymorphisms. Genotyping of the eNOS intron 4 polymorphisms was performed by PCR assay. Our results indicated that there was a significant difference in the frequencies of the genotypes between case and control groups for the polymorphisms in both intron 4 (odds ratio (OR) 5.09, 95 % confidence interval (CI) 2.091–12.396, P?<?0.001) and T-786C (OR 7.389, 95 % CI 3.043–17.942, P?<?0.001). In our study, the eNOS polymorphisms (in intron 4 and T-786C) were found to be significantly associated with increased risk of RSM in Egyptian women.