Folate receptor-mediated delivery system based on chitosan coated polymeric nanoparticles for combination therapy of breast cancer.

Combination therapy using two or more drugs with different mechanisms of action is an effective strategy for treating cancer. This is because of the synergistic effect of complementary drugs that enhances their effectiveness. However, this approach has some limitations, such as non-specific distribution of the drugs in the tumor and the occurrence of dose-dependent toxicity to healthy tissues. To overcome these issues, we have developed a folate receptor-mediated co-delivery system that improves the access of chemotherapy drugs to the tumor site. We prepared a nanoplatform by encapsulating paclitaxel (PTX) and curcumin (CUR) in poly(caprolactone)-poly(ethylene glycol)-poly(caprolactone) (PCL-PEG-PCL) co-polymer using a double emulsion method and coating nanoparticles with pH-responsive chitosan-folic acid (CS-FA) conjugate. The nanocarrier's physicochemical properties were studied, confirming successful preparation with appropriate size and morphology. PTX and CUR could be released synchronously in a controlled and acid-facilitated manner. The dual drug-loaded nanocarrier exhibited excellent anti-tumor efficiency in MDA-MB-231 cells in vitro. The active targeting effect of FA concluded from the high inhibitory effect of dual drug-loaded nanocarrier on MDA-MB-231 cells, which have overexpressed folate receptors on their surface, compared to Human umbilical vein endothelial cells (HUVEC). Overall, the nanoengineered folate receptor-mediated co-delivery system provides great potential for safe and effective cancer therapy.

The substance P/ neurokinin-1 receptor signaling pathway mediates metastasis in human colorectal SW480 cancer cells.

BACKGROUND: The substance P (SP)/neurokinin-1 receptor (NK1R) system, a critical metastatic signaling pathway, can be targeted by substance P antagonists to prevent its cancer-progressive impacts. In the current study, we aimed to investigate the carcinogenic activity of the SP/NK1R system in human SW480 colorectal cancer cells and study the antagonistic impact of aprepitant (AP) by measuring MMP-2 and MMP-9 enzymatic activity. METHODS: Different concentrations of SP, alone or mixed by AP, were utilized to treat SW480 cells to investigate the cells' viability and metastasis by applying Resazurin and Gelatin Zymography methods, respectively. The cells' metastatic response was analyzed by measuring the MMP-2 and MMP-9 in transcriptional and translational levels. Finally, the Scratch assay was carried out to evaluate the cells' metastatic response following the SP/AP treatment. RESULTS: A significant metastatic activity was observed in SW480 cells following incubation with the increasing SP doses by detecting MMP-2/MMP-9 enzyme activity, genes overexpression, and enhanced cell migration. This is while the AP treatment meaningfully diminished all the SP-mediated metastatic effects (p-Value < 0.001). CONCLUSIONS: According to the results, the SP/NKR1 signaling pathway can be considered one of the main metastatic effectors in human colorectal cancer. Therefore, AP might be suggested to be used as the SP antagonist and an efficient anti-metastatic drug.

A novel strategy for engineering of a smart generation of immune ribonucleases against EGFR+ cells.

The overexpression of epidermal growth factor receptor (EGFR) could result in the development of solid tumors of prostate, breast, gastric, colorectal, ovarian, and head and neck, leading to carcinoma. Antibody therapies are ideal methods to overcome malignant diseases. However, immunoribonucleases are a new generation of antibodies in which an RNase binds to a specific antibody and shows a stronger ability to terminate cancer cells. In this study, we engineered Rana pipiens RNase to bind to the scFv of human antiepidermal growth factor receptor antibody. The molecular dynamic simulations confirmed protein stability and the ability of scFv-ranpirnase (rantoxin) to bind to epidermal growth factor receptor protein. Then, the rantoxin construct was synthesized in a pCDNA 3.1 Neo vector. CHO-K1 cells were used as expression hosts and the construct was transfected. Cells were selected by antibiotic therapies using neomycin, 120 mg/ml, and the high-yield colony was screened by real-time polymerase chain reaction (PCR) methods. Then, the recombinant protein production was confirmed using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. The molecular dynamic simulation (MDS) confirmed that the I467, S468, Q408, and H409 amino acids of EGFR bonded well to rantoxin. As revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, the rantoxin production and PCR analysis showed that the T3 colony can produce rantoxin messenger RNA fourfold higher than the GAPDH gene. The immunotoxin function was assessed in A431 cancer cells and EGFR-negative HEK293 cells, and IC50  values were estimated to be 22.4 ± 3 and >620.4 ± 5 nM, respectively. The results indicated that the immunotoxins produced in this study had the potential for use as anticancer drugs.

Zinc and low-dose of cadmium protect sertoli cells against toxic-dose of cadmium: The role of metallothionein.

BACKGROUND: The impact of cadmium (Cd) on male infertility may be related to the interaction with metal-binding proteins known as metallothioneins (Mts). Trace elements like zinc (Zn) have protective effects on testicular damage induced by Cd. OBJECTIVE: We determined the effect of Zn and low-dose Cd pre-treatment on the expression of Mt1 and Mt2 genes on testicular Sertoli cells. MATERIALS AND METHODS: The cultured TM4 mouse sertoli cells were treated with 50 µM ZnSO4 (Zn pre-treated group; ZnPG), 2 µM CdCl2 (Cd pre-treated group; CdPG), or distilled water (DW pre-treated group; DWPG). After 18 hour, all of these groups were exposed to 100 µM CdCl2 for different periods of time (1, 2, 3, and 6 hours). There was also a control group for all three groups, which was treated only with distilled water (without Cd or Zn pre-treatment). Cellular viability, Zn and Cd concentrations and gene expression were assessed by MTT, atomic absorption spectrometry and real time PCR methods, respectively. RESULTS: The expression of Mt1 and Mt2 genes in ZnPG, CdPG, and DWPG was greater than the control group (p=0.02 and p=0.01, respectively). Cd concentrations in CdPG and DWPG were greater than the control group (p=0.00). Expression of both genes in ZnPG and CdPG increased after 3 hours of treatment and Cd concentration decreased simultaneously, which was more obvious in ZnPG. CONCLUSION: Zn and short term low-dose Cd pre-treatment might reduce the adverse effects of Cd by increasing expression of Mts genes in Sertoli cells. The protective effect of Zn was stronger than Cd.

Distribution of fatty acids in adipose tissue of patients with type 2 diabetes.

BACKGROUND: It is not known whether alterations of adipose tissue fatty acid compositions predict type 2 diabetes. Our goal was to characterize the relationship between the adipose tissue fatty acid compositions and type 2 diabetes. METHODS: We evaluated the fatty acid compositions of subcutaneous adipose tissue. These analyses were carried out on samples from 76 normal and 98 diabetic adults. Analysis was performed on a gas chromatograph. RESULTS: The adipose tissue palmitic acid composition of diabetic subjects (24.9% +/- 0.3) was significantly higher (p = 0.01) than in the controls (23.3% +/- 1.6). A significant negative correlation (r = -0.276, p = 0.001) was found between adipose tissue polyunsaturated fatty acids (PUFAs) and the Chol/HDL-C ratio of all the subjects studied (controls plus diabetic patients). A similar negative correlation (r = -0.429, p = 0.001) was identified only for control group, whereas no significant correlation (p > 0.05) was found for diabetic populations. The correlation between fatty acid compositions and serum lipid profiles (TG, HDL-C, Chol/HDL-C, LDL-C/HDL-C proportion) and fasting blood sugar (FBS) with HbAlc concentrations of people with diabetes and controls was not significant. CONCLUSIONS: A high amount of palmitic acid in adipose tissue may increase the risk of the type 2 diabetes and it seems that a high intake of PUFAs can lead to lowering of the Chol/HDL-C ratio.

Association of plasma nitric oxide concentration and endothelial nitric oxide synthase T-786C gene polymorphism in coronary artery disease.

Nitric oxide (NO) is synthesized from l-arginine by endothelium nitric oxide synthase (NOS3) and plays important roles in many physiologic and pathologic processes. NO involved in the pathogenesis of coronary atherosclerosis. In the present study we hypothesized that polymorphisms of NOS gene might be associated with increased risk of coronary artery disease (CAD) and plasma NO concentrations. The eNOS gene polymorphism was investigated in 241 unrelated CAD patients with positive coronary angiograms and 261 ages matched control subjects without a history of symptomatic CAD. The NOS3 gene polymorphisms were analyzed by RFLP. Plasma NO, lipid profile and other risk factors were also assessed. The genotype frequencies for T-786C polymorphism differed significantly between CAD patients and controls (p=0.041). The mean plasma NO(x) concentrations showed significant differences according to genotypes of T-786C polymorphism in total population only. The mean plasma NO(x) increased in those individuals that are homozygote for C allele in promoter compared with those individuals are heterozygote for this allele and homozygote for T allele in total population and Controls, but no in CAD patients. The present study provides evidences that T-786C polymorphism of the NOS3 gene is associated with CAD. T-786C polymorphism was not associated with increased plasma NO in CAD patients.

Fatty acid composition of serum lipids in patients with type 2 diabetes.

BACKGROUND: Serum fatty acid content mainly reflects dietary fat intake. It is not known whether alterations of serum fatty acid compositions predict type 2 diabetes risk. METHODS: We evaluated fatty acid contents in serum of the 76 normal and 98 diabetic adults taking part in a cross sectional study, using a gas chromatography method. RESULTS: The contents of total saturated (SFAs) and monounsaturated fatty acids (MUFAs) of the diabetic subjects were significantly higher when compared to the controls (p = 0.006, p = 0.02 respectively). Serum linoleic and polyunsaturated fatty acid (PUFAs) contents of subjects with diabetes were significantly lower than the control group (p = 0.02). There was a negative correlation (p = 0.001, r = -0.429) between serum PUFAs contents and cholesterol/HDL-C ratio in the control group, whereas this correlation in people with diabetes was not significant (p = 0.12). The correlation between serum TG (triglyceride) and SFAs contents of the diabetic patients was significantly positive (p = 0.01, r = 0.252). A significant negative correlation (p = 0.001, r = -0.421) was found between PUFA contents and serum TG levels of people with diabetes; however, the same correlation for the control group was not significant (p = 0.56). CONCLUSIONS: Diabetes incidence was significantly and positively associated with the proportions of serum palmitic, total saturated and monounsaturated fatty acid contents. Our findings with the use of this biomarker suggest that the dietary fat profile, particularly that of saturated and monounsaturated fatty acids, may contribute to the etiology of diabetes. It seems that patients with type 2 diabetes can have good control on lipid parameters with a higher intake of polyunsaturated fatty acids than saturated fatty acids.

Effect of contraceptive pills on the activity status of the antioxidant enzymes glutathione peroxidase and superoxide dismutase in healthy subjects.

BACKGROUND: Experimental evidences suggest that metabolic activation and conversion of oral contraceptive pills (OCPs) to reactive species are responsible for their genotoxicity. The present study was undertaken to investigate the effects of low-dose (LD) OCPs on the activities of erythrocyte antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD) in OCP consumers. STUDY DESIGN: Enzyme activities were assayed spectrophotometrically in 50 healthy women with normal menstrual cycles who served as the control group and 50 women taking LD OCPs. RESULTS: The pooled data obtained for erythrocyte GPX activity in OCP consumers (50.05 ± 14.9 U/g of Hb) was significantly higher (+15.4%, p = .015) than in the control group (42.33 ± 16.31 U/g of Hb), but the same comparison for SOD activity between the control group (83.46 ± 23.97 U/g of Hb) and women receiving OCPs (81.83 ± 23.97 U/g of Hb) showed an insignificant (-2%, p = .699) decrease. The duration of intake of OCPs beyond 36 months had an effect on the magnitude of the increase (+16.2%) and the decrease (-11%) in GPx and SOD activities, respectively. There was a significant and considerable (not significant) correlation between the activities of GPx (p = .039) and SOD (p = .102) with the duration of OCP consumption, respectively. CONCLUSION: These findings suggested that OCPs may stimulate or reduce the activities of GPx and SOD enzymes, respectively. This may be due to an effect of these pills on bone marrow erythroblast maturation via stimulation or inhibition of the synthesis of new active GPx and SOD molecules or may be a result of the increased frequency of an allele of the GPx and SOD enzymes. It is suggested that these alterations in GPx and SOD activities may be related to their probable protective effects in response to various pathological and physiological properties of OCPs. It seems that probably free radicals produced during metabolism of OCPs provoke the activity of antioxidant enzymes.

Effect of apolipoprotein E genotypes on incidence and development of coronary stenosis in Iranian patients with coronary artery disease.

BACKGROUND: Apolipoprotein (apo) E polymorphism plays a significant role in the development of coronary disease, but their involvement in coronary artery stenosis (CAS) is controversial. Therefore, the purpose of this study was to investigate the effects of this polymorphism on atherosclerosis, and severity and extent of CAS in unrelated Iranian population. METHODS: DNA was isolated from 390 study participants and APOE genotypes were determined utilizing the polymerase chain reaction and restriction fragment length polymorphism. RESULTS: The APOE-ε4 and -ε2 allele frequencies were significantly higher in the CAS patients than in the control group (P<0.05). The association of Apo E polymorphism with the severity of stenosis was evaluated, which is according to the result that apolipoprotein E alleles were not significantly different when compared with the severity of stenosis (χ(2) =0.84, P>0.05). CONCLUSION: Our results suggest that APOE-ε4 is a risk factor for stenosis but does not has any effect on the severity of this disease.

SstI Polymorphism of the Apolipoprotein CIII Gene in Iranian Hyperlipidemic Patients: A Study in Semnan Province.

OBJECTIVES: The Sst-I polymorphic site on the 3' untranslated region of the apo CIII gene, has been previously reported to be associated with hypertriglyceridemia. The aim of the present study was to explore the association between Sst-I polymorphism with plasma lipid and lipoprotein levels in hyperlipidemic (HLP) patients from Semnan province, Iran. MATERIALS AND METHODS: Genomic DNA was prepared from 76 patients with HLP and 75 matched healthy subjects. DNA samples were amplified by polymerase chain reaction. The samples were analyzed by restriction fragment length polymorphism (RFLP) method using SstI enzyme. RESULTS: The genotype and allelic frequencies for this polymorphism were significantly different between HLP and normolipidemic groups (P< 0.002). Plasma triglyceride (TG) level was higher in both groups, in S2S2 genotype was more than in the S1S1and S1S2 genotypes, however, there was no significant difference in comparison with the control group. Subjects with S1S2 + S2S2 genotypes in compare to S1S1 genotype had odd ratio of 2.8 (95% CI: 1.41-5.56, P< 0.003) for developing hypertriglyceridemia. CONCLUSION: The results showed that the presence of rare S2 allele was associated with change in TG level in the selected population.

Matrix metalloproteinase-9 and paraoxonase 1 Q/R192 gene polymorphisms and the risk of coronary artery stenosis in Iranian subjects.

PURPOSE: To investigate the association of matrix metalloproteinase-9 (MMP-9) and paraoxonase 1 (PON1) 192 polymorphisms with susceptibility to coronary artery stenosis (CAS) and the number of diseased vessels in patients with CAS. METHODS: The study population comprised 302 unrelated Iranian individuals, including 145 patients with CAS and 157 control subjects. Genotypes for MMP-9 and PON1 192 polymorphisms were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). RESULTS: In our study, distributions of the TT genotype of MMP-9 and the RR genotype of PON1 192 were significantly higher in patients compared with healthy control subjects (P<0.05). Subsequent analysis demonstrated that a significant difference existed in the male (TT+TC vs. CC and RR+QR vs. QQ, P<0.01) but not in the female. The associations of these polymorphisms with the severity of stenosis were also evaluated, which according to results distribution of MMP-9 and PON1 192 genotypes were not significantly different compared with the severity of stenosis (P>0.05). CONCLUSIONS: The observation indicates that the polymorphisms in the MMP-9 and PON1 192 genes potentially play a role in the manifestation of coronary atherosclerosis but does not have any effect on the number of diseased vessels in Iran.

Differential gene-expression of metallothionein 1M and 1G in response to zinc in sertoli TM4 cells.

BACKGROUND: Zinc (Zn) as an important trace element is essential for testicular development and spermatogenesis. Molecular mechanism of Zn action in the reproductive system may be related to metal binding low-molecular weight proteins, metallothioneins (MT). Our objective was to determine the effect of Zn on two important isoforms of MT, MT1M and MT1G genes expression on testicular sertoli cells. METHODS: Cultured sertoli TM4 cells were exposed to different concentrations of Zn at different time points. Cellular uptake of Zn was tested using flame atomic absorption spectrometry. The cellular viability and gene expression were assessed by MTT and real-time PCR methods, respectively. RESULTS: The treated cells resulted in higher Zn concentration and cellular viability. The expression of MT1M and MT1G genes in the treated cells were greater than those of the untreated cells (P less than 0.05). In the high dosage treated group (100 and 500 muM), Zn concentration and expression of MT1M and MT1G genes increased three h after treatment; MT1G gene expression increased more at sixth h. At 18th h of treatment, the expression of both genes especially MT1G, increased dramatically while Zn concentration decreased. CONCLUSION: Since the increase of MT1G mRNA was coincident with cellular Zn level, it seems that MT1G has a more prominent role than MT1M in the homeostasis of Zn. In addition, Zn at dosage of 50 muM (pharmacologic concentration) may protect cells by increasing the expression of MT genes at longer periods.

Endothelial nitric oxide synthase gene Glu298Asp polymorphism in patients with coronary artery disease.

BACKGROUND AND OBJECTIVES: Endo-derived nitric oxide (NO) is synthesized from L-arginine by endothelial nitric oxide synthase (NOS3). Since reduced NO synthesis in endothelial cells has been implicated in the development of coronary atherosclerosis, we investigated the association of NOS3 gene polymorphisms and coronary artery disease (CAD) in an Iranian population. SUBJECTS AND METHODS: We studied the NOS3 gene Glu298Asp polymorphism in 241 CAD patients with positive coronary angiograms (i.e.,> 50% stenosis affecting at least one coronary vessel) in Shahid Rajaee Heart Hospital and 261 control subjects without a history of symptomatic CAD. The NOS3 gene polymorphism was analyzed by polymerase chain reaction and restriction fragment length polymorphism. Lipid profile and other risk factors were also determined. RESULTS: The genotype frequencies of Glu298Asp polymorphism for Glu/Glu, Glu/Asp, and Asp/Asp were 61.3%, 32.2%, and 6.5%, respectively, in control subjects, and 46.5%, 42.7%, and 10.8% in CAD patients, respectively. The genotype frequencies differed significantly between the two groups (P=.003). The frequencies of the Asp alleles were 32.2% and 22.6% for CAD patients and control subjects, respectively; the difference between the two groups was statistically significant (P=.001; odds ratio=1.6). Plasma lipids, except HDL-C, were also significantly increased in the CAD groups. CONCLUSION: These results suggest that CAD is associated with Glu298Asp polymorphism of the NOS3 gene in our population and that this polymorphism is an independent risk factor for CAD.

Angiotensin converting enzyme DD genotype not associated with increased risk of coronary artery disease in the Iranian population.

The coronary artery disease (CAD) is of the main causes of heart failure and there is evidence supporting the association of angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and the susceptibility to CAD. Therefore, the relevance of ACE polymorphism for CAD was determined in the Iranian population. 487 age-matched individuals including 224 patients with >50% angiographically established coronary stenosis and 263 healthy subjects genotyped for ACE gene I/D polymorphism by a standard method. Paraclinical characteristics including lipid profile were also determined for both groups. While the systolic (p<0.0001) and diastolic (p<0.0001) blood pressure, serum cholesterol (p<0.005) and LDL-C (p<0.05) were significantly increased in CAD patients, our results show that there was no increased risk of CAD in association with DD genotype in Iranian population. Allele frequencies were also similar in both groups. Although, we found a significant difference in ID (p<0.005) and II (p<0.05) genotype between patients and healthy subjects. The present study showed that DD genotype does not increase the CAD susceptibility in the studied Iranian population and may not be as a risk factor. Therefore, further studies together with the other polymorphisms of ACE gene may be required to determine the relation between cardiovascular disease susceptibility and ACE genetic variations in Iranian population.

Angiotensin II Differentially Induces Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 Production and Disturbs MMP/TIMP Balance.

Angiotensin II, the main component of the renin-angiotensin system, is associated with cardiovascular diseases such as hypertension, vascular remodeling and inflammation. Remodeling process results from dysregulation of Matrix Metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). MMPs are considered as important target genes for angiotensin II. The aim of this study was to determine the effects of angiotensin II on MMP-9 and TIMP-1 production and MMP/TIMP balance in a monocytic cell type. Human monocytic U-937 cells were cultured and treated with 100 nM angiotensin II. Supernatants were analyzed for MMP-9 and TIMP-1 using ELISA and zymography methods. Real-time PCR was utilized to evaluate relative MMP-9 and TIMP-1 genes expression following treatments. Cytotoxicity potentials of treatments were determined by assaying lactate dehydrogenase leakage from the cells. Stimulation of the monocytic cells with angiotensin II significantly increased MMP-9 and TIMP-1 secretion as measured by ELISA (p < 0.05). It also augmented gelatinolytic activity of MMP-9 in the conditioned media as much as 49% (p < 0.05). Incubation of the cells with angiotensin II for 12 hr increased MMP-9 and TIMP-1 gene expression 2.7 and 1.8 folds, respectively (p < 0.05). Angiotensin II treatments did not establish significant cytotoxic effects. In summary, our data provide further evidences that monocytic MMP-9 is a major effector of angiotensin II. It is induced more efficiently than TIMP-1 by angiotensin II that leads to MMP/TIMP imbalance. Our data also reveal the pivotal participation of these cells in pathological cardiovascular remodeling mediated by angiotensin II.

Effect of contraceptive pill on the selenium and zinc status of healthy subjects.

BACKGROUND: The study was conducted to ascertain the influence of oral contraceptive pill (OCP) uptake on serum zinc and selenium in contraceptive pill users. STUDY DESIGN: The concentration of zinc and selenium was determined by atomic absorption spectrophotometer in 50 healthy women with normal menstrual cycles as a control group and 50 women taking low-dose OCP. RESULTS: The control reference values were 81.61+/-9.44 and 70.35+/-25.57 mcg/dL, which were obtained for zinc and selenium, respectively. Use of OCP resulted in a significant decrease in serum zinc levels (p

Taq1B polymorphism of cholesteryl ester transfer protein (CETP) gene in primary combined hyperlipidaemia.

BACKGROUND & OBJECTIVE: Cholesteryl ester transfer protein (CETP) gene polymorphism is known to be associated with changes in lipid profiles. Primary hyperlipidaemia is considered to be a major risk factor for pancreatitis, atherosclerosis and coronary heart disease. We investigated the association of one common polymorphism in the CETP gene (Taq1B) with plasma lipid levels and CETP activity in Iranian subjects with and without primary combined hyperlipidaemia. METHODS: The study included 102 patients with primary combined hyperlipidaemia and 214 health individuals. Polymerase chain reaction and restriction fragment length polymorphisms were used for genotype detection. To determine the relationship between Taq1B polymorphism and lipid levels, lipids and CETP activity were measured in primary combined hyperlipidaemic and normolipidaemic subjects, with and without Taq1B polymorphism. RESULTS: Plasma CETP activity was significantly (P<0.001) higher in primary combined hyperlipidaemic individuals than in controls. Plasma HDL-C was higher in both groups, in the B(2)B(2) genotype than in the B(1)B(1) and B(1)B(2) genotypes, whereas the serum TG concentrations and CETP activity were lower in B(2)B(2) genotype compared with other genotypes (B(1)B(1) and B(1)B(2)). The genotype and allelic frequencies for this polymorphism differed significantly between hyperlipidaemic and nonmolipidaemic individuals (P<0.05). In both groups, CETP Taq 1B polymorphism (presence of B(2) allele) correlated significantly with HDL-cholesterol (HDL-C) (r=0.201 and r=0.452 in control and patient groups respectively) and CETP activity (r= -0.123 for controls and r= -0.192 for patients). INTERPRETATION & CONCLUSION: The results showed that Taq 1B polymorphism of CETP gene was associated with changes in lipids profile and plasma CETP activity in the selected population and might have a role in contributing to genetic risk of developing coronary artery disease.

Influence of genetic polymorphism in matrix metalloproteinase-3 on extent of coronary atherosclerosis and risk of coronary artery stenosis.

BACKGROUND AND AIMS: Matrix metalloproteinase-3 (MMP3) is key member of the MMP family. It is known to be present in coronary atherosclerosis. Several studies have demonstrated that MMP-3 5A/6A polymorphism modifies each transcriptional activity in an allele-specific manner. We hypothesized that this polymorphism may be a risk factor for the development of coronary artery stenosis (CAS). We estimated the effect of MMP3 (5A/6A) gene polymorphism on CAS risk in an Iranian population. METHODS: One hundred ninety patients with CAS and 200 healthy controls were in this study. MMP3 genotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). RESULTS: Significant differences between cases and controls were observed for MMP3 genotype frequencies (chi(2)=199.305, p<0.001). The 6A allele was less frequently seen in the control group compared with the disease group (85.79 vs. 78%, 6A/6A+5A/6A vs. 5A/5A, p< or =0.05). Association of this polymorphism with CAS severity was evaluated in the two groups, and distribution of the MMP3 genotype was not significantly different as compared with CAS severity (p>0.05). CONCLUSIONS: These data imply involvement of the -1612 5A/6A polymorphism in CAS and also that the 6A/6A MMP-3 genotype is a genetic susceptibility factor for CAS (but does not affect disease severity).

Paraoxonase 1 gene promoter polymorphisms are associated with the extent of stenosis in coronary arteries.

HDL-associated paraoxonase1 (PON1) is believed to be an important anti-oxidative enzyme in the retardation of atherosclerosis. In this study, we determined haplotypes of three SNPs within the PON1 gene promoter to elucidate association of functional sites with coronary artery disease (CAD). We applied a direct haplotyping procedure through ARMS (Amplification Refractory Mutation System) and RFLP (Restriction Fragment Length Polymorphism) analysis techniques. The haplotypes of the G(-907)C, A(-162)G and C(-107)T polymorphisms within the 5' region of the PON1 gene were determined in 99 patients and 66 controls who were evaluated angiographically for the presence and extent of stenosis in coronary arteries. The genotype and haplotype distributions had significant differences between patient subgroups (One-, Two- and Three-vessel disease) but not between the patient and control groups. Multivariate analyses suggested decreased arylesterase activity is the most important independent factor in the CAD severity. The increase of high activity variants [G(-907) and C(-107)] within the two-allelic haplotypes was reversely associated with the extent of stenosis in coronary arteries. However, we could not determine the independent involvement each of the C(-107)T and G(-907)C polymorphisms on the extent of stenosis. We found no significant association between the A(-162)G polymorphism and the extent of stenosis in vessels. The study indicated the association of polymorphic variations within the PON1 gene promoter haplotypes with the serum arlyesterase activity. The arlyesterase activity was also associated with the extent of stenosis in coronary arteries but not with primary development of atherosclerosis.

Lack of evidence for contribution of intron4a/b polymorphism of endothelial nitric oxide synthase (NOS3) gene to plasma nitric oxide levels.

OBJECTIVE: Nitric oxide (NO) is synthesized from L-arginine by endothelium nitric oxide synthase (NOS3). It has been shown that reduced NO synthesis in endothelial cells has been implicated in the development of coronary atherosclerosis. We hypothesized that polymorphisms of NOS3 gene might be associated with increased susceptibility of coronary artery disease (CAD) and plasma NO concentrations. METHODS AND RESULTS: We studied the NOS3 intron4 gene polymorphism in 141 unrelated CAD patients with positive coronary angiograms in the Shahid Rajaee Heart Hospital and 159 age-matched control subjects without a history of symptomatic CAD. The NOS3 gene polymorphism was analysed by polymerase chain reaction. Plasma NO was also determined. The genotype frequencies for NOS34a/b polymorphism differed significantly between CAD patients and controls (P = 0.041). Mean plasma NOx concentration was significantly higher (P = 0.0009) in CAD patients (87.5 microM) than in controls (62.7 microM). Mean plasma NOx concentrations in the subjects with 4a allele and in the subjects without 4a allele were not significantly different. Plasma lipids, except HDL-C were also remarkably increased in the CAD group. CONCLUSIONS: The present study provides evidence that the intron4a/b polymorphism of the NOS3 gene is associated with CAD. Mean plasma NO was higher in CAD patients than in control subjects. The NOS34a/b polymorphism was not associated with increased plasma NO.