The effect of adiponectin on matrix metalloproteinase-9 (MMP-9) in vascular smooth muscle cells

Background & Aims: Atherosclerosis is a major cause of morbidity and mortality. Adiponectin reduces the risk of heart disease, and matrix metalloproteinase-9 (MMP-9) is involved in the formation and development of atherosclerotic plaque. The aim of this study was the investigation of the effect of adiponectin on MMP-9 gene expression. It seems this hormone can reduce the risk of atherosclerosis by MMP-9 gene expression reduction. Methods: Human aorta smooth muscle cells were cultured in F12K media in appropriate environment and conditions, then, the cells were treated with 5 µg/ml adiponectin. After 24 and 48 hour, total RNA was extracted and corresponding cDNA was made. After drawing standard curve and determining the efficiency of the reaction, MMP-9 gene expression was measured by the SYBR Green kit and real time PCR method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was the reference gene. The amount of MMP-9 protein, compared to the β-actin protein as reference protein, was estimated with Western blot method. Results: adiponectin did not cause a ch ange in gene expression of MMP-9 in 24 hours, but in 48 hours reduced gene expression (-1.1, and -5.5, respectively). As a result of MMP-9 gene expression reduction, protein also reduced after 48 hours compared to β-actin protein. Conclusion: Through the reduction of MMP-9 protein and gene expression, adiponectin changes extra cellular matrix which may reduce the risk and complications of atherosclerosis. © 2014, Kerman University of Medical Sciences. All rights reserved

Adiponectin inhibits oxidized low density lipoprotein-induced increase in matrix metalloproteinase 9 expression in vascular smooth muscle cells

BACKGROUND: High expression of matrix metalloproteinase 9 (MMP9) during vascular injury and inflammation plays an important role in atherosclerotic plaque formation and rupture. In the process of atherosclerosis, oxidized low-density lipoprotein (oxLDL) upregulates MMP9 in human aortic vascular smooth muscle cells (HA/VSMCs). Adiponectin is an adipose tissue-derived hormone that has been shown to exert anti-atherogenic and anti-inflammatory effects. The aim of this study was to investigate the effect of adiponectin on MMP9 expression under pathogenic condition created by oxLDL in HA/VSMCs. METHODS: In this experimental study, HA/VSMC were stimulated with oxLDL alone and in the presence of adiponectin for 24 and 48 h. The expression of MMP9 gene was determined by real-time polymerase chain reaction method. The protein level of this gene was investigated by western blotting technique. RESULTS: An oxLDL increased MMP9 expression 2.16 +/- 0.24- and 3.32 +/- 0.25-fold after 24 and 48 h, respectively and adiponectin decreased oxLDL-induced MMP9 expression in a time-dependent manner. CONCLUSION: These results show that adiponectin changes extracellular matrix by reducing MMP9 mRNA and protein, therefore, may stabilize lesions and reduce atheroma rupture

Effects of Feeder Layers, Culture Media, Conditional Media, Growth Factors, and Passages Number on Stem Cell Optimization

Stem cells are undifferentiated and self-renewal cells which could be obtained from the body or artificially derived from an adult somatic cell by forced expression of specific genes. In recent years stem cells are widely used in laboratory for tissue engineering and therapeutic applications. There are different factors and conditions that affect the stem cell culture such as feeder layers, atmosphere, kind of medium, growth factors, passages number, and conditional media with animal or human sources. Optimization of stem cell culture for medical approaches and regenerative medicine is important. Therefore, in the present study, the effect of these factors and agents on optimization of stem cell culture has been discussed. This review study showed that optimization of feeder layer, atmosphere, and using supplemented media with essential growth factors could help in maintaining the stem cells in undifferentiated state in vitro. The present study indicated that optimization of stem cell culture depends on the kind of each cell type and using stem cells in low passage number could decrease chromosomal abnormalities and DNA damages. For inhibiting the stem cell contamination by feeder cell lines, culture of these cells on feeder free systems like Matrigel matrix in conditioned media supplemented with essential growth factors is useful. Also, for eradicating immune system responses and reducing the risk of animal pathogen transfer, culture of stem cells on human feeder in optimized media is suitable for therapeutic approaches and regenerative medicine

Effects of Phenanthrene and Pyrene on Cytogenetic Stability of Human Dermal Fibroblasts Using Alkaline Comet Assay Technique

In the present study, the influence of phenanthrene and pyrene on cytogenetic stability of human dermal fibroblasts using alkaline comet assay was evaluated. The cells were isolated from foreskin samples obtained from healthy male infants and in low passages (P 1–3) were exposed to various concentrations (0.0900, 0.0625, 0.0320, and 0.0066 ppm) of phenanthrene and pyrene. Then, alkaline comet assay was performed to investigate the influence of these compounds on DNA damage and cytogenetic stability of human dermal fibroblasts. In the present work H2O2 treatment was used as a positive control of comet assay to create DNA damages. The analysis of alkaline comet assay parameters by CaspLab software showed the tail DNA% in high concentration (0.0900 ppm) of phenanthrene and pyrene in the exposed cells got increased as compared to normal cells, while head DNA% decreased. Also, similar to positive control (H2O2), DNA damage with long tail comet was observed in high concentration of these compounds as compared to normal cells. The comparison of comet assay parameters specially head DNA% and tail DNA% between each concentrations of phenanthrene and pyrene with other groups (healthy cells and H2O2 treatment) by ANOVA and post hoc Tukey test showed that these parameters were more significantly different (p < 0.05). These results indicated that phenanthrene and pyrene even in low dosage are dangerous and can increase the DNA damage and affect on cytogenetic stability of dermal fibroblasts. These findings suggested that phenanthrene and pyrene could increase the related diseases and risk of skin cancer in exposed individuals

Compound Heterozygosity for Two Novel SLC26A4 Mutations in a Large Iranian Pedigree with Pendred Syndrome

Objectives. The aim of this study was to detect the genetic cause of deafness in a large Iranian family. Due to the importance of SLC26A4 in causing hearing lots, information about the gene mutations can be beneficial in molecular detection and management of deaf patients. Methods. We investigated the genetic etiology in a large consanguineous family with 9 deaf patients from Ban province of Iran with no GJB2 mutations. Initially, linkage analysis was performed by four DFNB4 short tandem repeat markers. The result showed linkage to DFNB4 locus. Following that, DNA sequencing of all 21 exons, their adjacent intronic sequences and the promoter of SLC26A4 was carried out for mutation detection. Results. Two novel mutations (c.863-864insT and c.881-882delAC) were identified in exon 7 of the gene, in both homozygous and compound heterozygous state it patients. Conclusion. Our results supported the importance of the SLC26A4 mutations in the etiology of hearing loss among the Iranian patients and therefore its mutation screening should be considered after GJB2 in the molecular diagnostics of hearing loss, especially when enlarged vestibular aqueduct or goiter is detected

Expression and Purification of the Recombinant Cytochrome P450 CYP141 Protein of Mycobacterium Tuberculosis as a Diagnostic Tool and Vaccine Production

Background: Tuberculosis (TB) is regarded as a health problem worldwide, particularly in developing countries. Mycobacterium tuberculosis (M. tuberculosis) is the cause of this disease. Approximately two billion people worldwide are infected by M. tuberculosis and annually about two million individuals die in consequence. Forty million people are estimated to die because of M. tuberculosis over the next 25 years if the measures for controlling this infection are not extensively developed. In the vaccination field, Bacillus Calmette-Guerin (BCG) is still the most effective vaccine but it shows no efficacy in adult pulmonary patients. One of the other problems regarding TB is its appropriate diagnosis. Objectives: In this experimental study, the recombinant cytochrome P450 CYP141 protein of M. tuberculosis was expressed and purified to be used as a vaccine candidate and diagnostic purpose in subsequent investigations. Materials and Methods: The optimization of the cytochrome P450 CYP141 protein expression was evaluated in different conditions. Then, this protein was purified with a resin column of nickel-nitrilotriacetic acid and investigated via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting. Results: The highest expression of the cytochrome P450 CYP141 protein was obtained by the addition of 1 mM of isopropyl beta-D-1-thiogalactopyranoside (IPTG) to the bacterial culture grown to an optical density at 600 nm (OD600) of 0.6, 16 hours after induction. This protein was subsequently purified with a purification of higher than 80%. The results of Western Blotting indicated that the purified protein was specifically detected. Conclusions: In this experimental study, for the first time in Iran the expression and purification of this recombinant protein was done successfully. This recombinant protein could be used as a vaccine candidate and diagnostic purpose in subsequent investigations

Cytogentic analysis of human dermal fibroblasts (HDFs) in early and late passages using both karyotyping and comet assay techniques

Human dermal fibroblasts (HDFs) are a potential source of somatic cells for genetic manipulation and tissue engineering. Confirmation of cytogenetic stability of these cells is an essential step for cell nuclear transfer and generation of a suitable and functional induced pluripotent stem cells line. HDF cells were isolated and cultured from human foreskin samples. Cytogenetic stability of these cells was evaluated in early (3-4) and late (10-15) passages using karyotype test and alkaline comet assay techniques. HDF cells in early and late passages showed normal karyotype but by comet assay abnormality and DNA damages in late passages of HDFs were observed. Also, the parameters of alkaline comet assay in early passages of HDFs compared with late passages and positive control groups more significantly were different (p < 0.05). These findings indicate that single-strand breaks or DNA damage after many passages may have occurred in HDF cells. Our results demonstrate that only early passages of HDF cells maintain cytogenetic stability and are good candidates for gene reprogramming. In conclusion, karyotype testing alone can not be used for detection of all signs of cytogenetic abnormality and DNA damages of cells. So, for precise evaluation of DNA damage and cytogenetic instability of fibroblast cells comet assay and karyotype techniques could complement each other

Effects of lentiviral vectors on DNA damage of human dermal fibroblasts (HDFs)

In present study we evaluated the DNA damages and cytogenetic stability of transducted and non-transducted human dermal fibroblasts (HDFs) by enhanced green fluorescent protein (eGFP) lentiviral vector using karyotyping, comet assay, and molecular techniques. HDFs were isolated from human foreskin samples and eGFP-expressing lentiviral vector were transfected into HEK-293T cells to produce lentiviruses. Then, HDFs at passage 2 were transducted with concentrated eGFP lentivirus and transducted HDFs were detected by fluorescent microscope. The expression levels of cell cycle genes include two subunits of anaphase promoting complex (APC) in transducted and nontransducted HDFs were measured by quantitative real-time PCR and finally, karyotype test and comet assay was performed to evaluate the DNA damages and cytogenetic stability in both groups. The results of karyotype analysis were not showed any abnormalities in karyotype of transducted HDFs by eGFP in compared to normal cells. The mean values of alkaline comet assay parameters on non-transducted (normal cells), eGFP-transducted group and positive control (H2O2 treatment) were calculated by CaspLab software. The comparison of mean difference of comet assay parameters include tail length, comet length, tail moment, and Olive tail moment by T test between eGFP-transducted HDFs and other groups (positive control and non-transducted HDFs) were statistically significant (p≤0.05). The alkaline comet assay on HDFs in eGFP-transducted group was showed small tail and indicated slight genetic damage compared with non-transducted group. Furthermore, the analysis of real-time PCR on expression of APC2 and APC7 genes in non-transducted HDFs compared with eGFP-transducted HDFs were not significant (p≤0.05). These findings indicated that integration of lentiviral vectors in first passage of transducted HDFs could not disturb the DNA structure and create chromosome instability. So in genetic engineering and gene transformation these vectors in first passages are useful

A study of cytogenetic stability of induced pluripotent stem cells using karyotyping and comet assay techniques

Background & Aims: Induced pluripotent stem cells (iPSCs) have the capability to undergo unlimited selfrenewal and differentiation into all cell types in the body. These cells are artificially derived from a nonpluripotent cell, typically human dermal fibroblasts (HDFs). The study of cytogenetic stability of these cells, in order to use iPS cells and apply studies in therapeutic applications, is essential. Methods: In the present experimental study, HDFs were isolated and cultured from human foreskin samples. The cytogenetic stability of these cells was evaluated in early passages (1-3) of HDFs using karyotype test and alkaline comet assay technique. The HDF cells treatment with hydrogen peroxide (H2O2) was used as a positive control for alkaline comet assay. The iPS cells with low passage (4-7) derived from reprogrammed HDFs were cultured on mouse embryonic fibroblast (MEF) feeder layer and cytogenetic stability of these cells were evaluated in early passages using karyotype test and alkaline comet assay technique. Results: The iPS cells in early passages (4-7) had normal karyotype (46, XY) and DNA damage and comet were not observed in these cells. In addition, HDF cells showed normal karyotype in early passages (1-3), but using comet assay, abnormality and DNA damages were observed in positive control (HDFs treated with H2O2). The comparison of alkaline comet assay parameters of iPS and HDF cells with positive control group showed statistically significant differences (P < 0.05). Conclusion: Since the comet assay is a sensitive technique for finding DNA damage, it is best if cytogenetic stability of these cells were evaluated before performing functional experiments on iPS cells. Therefore, for the precise evaluation of DNA damage and cytogenetic stability of iPS cells, the two techniques could complement each other. © 2015, Kerman University of Medical Sciences. All rights reserved

The assessment of lentiviral vectors application for gene transformation in human dermal fibroblasts (HDFs)

چکیده: زمینه و هدف: سلول های بنیادی پرتوان القایی Induced pluripotent stem cells= iPSc))، سلول های اولیه و تمایز نیافته ای هستند که قادر به ایجاد تقریباً هر نوع سلولی در بدن می باشند. هدف از پژوهش حاضر تولید و استفاده مؤثر از وکتور لنتی ویروسی TetO-FUW-OSKM جهت انتقال ژن ها به سلول های فیبروبلاست انسانی ((HDFs= Human fibroblast cells و در نهایت ارزیابی عملکرد این وکتور بود. روش بررسی: در این مطالعه تجربی پس از جداسازی و کشت سلول های HDF، وکتور لنتی ویروسی TetO-FUW-OSKM (به عنوان پلاسمید انتقال دهنده) حاوی ژن های برنامه ریزی مجدد همراه با پلاسمیدهای PsPAX2 و PMDG2 (به عنوان پلاسمیدهای کمکی لازم برای بسته بندی ویروس) به لاین سلولی HEK-293T جهت تولید ویروس ها ترانسفکت شدند. محیط رویی سلول های HEK-293T حاوی ویروس های تولید شده پس از 48 و 72 ساعت برداشت شد و این ویروس ها جهت برنامه ریزی مجدد سلول های HDF به این سلول ها ترانس داکت گردیدند. یافته ها: نتایج این مطالعه نشان دهنده تولید موفقیت آمیز وکتور لنتی ویروسی TetO-FUW-OSKM، کارایی مؤثر روش انتقال ژن با استفاده از وکتورهای لنتی ویروسی و بیان مناسب فاکتورهای رونویسی در سلول های HDF پس از ترانس داکشن بود. نتیجه گیری: با توجه به این یافته ها می توان از وکتورهای لنتی ویروسی جهت انتقال ژن و برنامه ریزی مجدد سلول های بالغ از قبیل HDF در مطالعات بعدی و تولید سلول های iPS استفاده کرد