Resveratrol reduced the detrimental effects of malondialdehyde on human endothelial cells.

Introduction: According to the statistics, vascular injury occurs during the onset of diabetic changes after the production of several byproducts. Many authorities have focused to find an alternative therapy for diabetic patients. In this study, we investigated the therapeutic effects of natural polyphenol like resveratrol on human endothelial cells exposed to malondialdehyde for 48 hours. Methods: Human Umbilical Vein Endothelial Cells were randomly classified into four groups;control, malondialdehyde (2.5 mM), resveratrol (100 µM), and cells received the combined regime for 48 hours. Cell viability was determined by 3-(4, 5-dimethyl thiazol-2-yl) 2, 5-diphenyl-tetrazoliumbromide (MTT) assay. Griess reaction was performed to measure the content of Nitric oxide (NO).Apoptosis was studied by using real-time polymerase chain reaction (RT-PCR) and western blotting assays. Levels of receptor tyrosine kinases like VEGFR-1, -2, Tie-1, and -2 were analyzed by enzyme-linked immunosorbent assay(ELISA). The affinity of resveratrol and malondialdehyde to serum albumin was measured by Surface Plasmon Resonance Assay. Any changes in chromatin remodeling were detected by PCR array analysis. Results: Resveratrol reduced cytotoxicity and NO content inside cells induced by malondialdehyde(MDA) (P < 0.05). Endothelial cell apoptosis was decreased by the reduction of pro-apoptotic factor Bax and increase of Bcl-2 following the incubation with resveratrol (P < 0.05). MDA-induced receptor tyrosine kinases increase was inhibited by resveratrol and reached near-to-normal levels (P < 0.05).Surface Plasmon Resonance revealed a higher affinity of resveratrol to albumin compared to the malondialdehyde-albumin complex. Polymerase chain reaction (PCR) array revealed the potency of resveratrol in chromatin remodeling following the treatment with malondialdehyde (P < 0.05). Conclusion: Based on our findings, resveratrol has the potential to decrease diabetic vascular injury induced by lipid byproducts such as MDA.

Downregulation of TdT Expression through Splicing Modulation by Antisense Peptide Nucleic Acid (PNA).

BACKGROUND AND OBJECTIVE: Antisense oligonucleotides are able to modulate splicing patterns and offer therapeutic intervention for cancer and other diseases. Considering TdT potential as a target in cancer therapy, the present study aimed to investigate splicing alteration of TdT pre-mRNA in Molt-4 cells using peptide nucleic acid (PNA) octaarginine and cholic acid conjugates. METHOD: We examined 16 mer PNAs targeting 5' and 3' junctions of intron 7 and addressed their mRNA splicing modulation effects using RT-PCR analysis. We also tested corresponding 2-base mismatch PNAs to confirm the sequence specificity. In addition, protien level of TdT, apoptosis induction and cell viability rate were analysed. RESULTS: PCR analysis showed that full match PNAs could modulate the splicing process, thereby producing a longer mRNA still including intron 7. PCR results also implied exon 7 skipping. In addition, reduced level of TdT protein in Molt-4 cells was observed. Downregulation of TdT level in PNA treated cells was accompanied by an increased rate of apoptosis and decreased the level of cell survival. CONCLUSION: PNA-mediated splicing modulation can specifically downregulate TdT expression. TdT dowregulation results in apoptosis induction and reduced cell survival in Molt-4 cells. These observations could draw more attentions to develop PNA based strategies for TdT suppression and consequent apoptosis induction in acute lymphoblastic leukemia.

Association of ACE, VEGF and CCL2 gene polymorphisms with Henoch-Schönlein purpura and an evaluation of the possible interaction effects of these loci in HSP patients.

BACKGROUND: Henoch-Schönlein purpura (HSP) is a multisystem, small vessel, leucocytoclastic vasculitis. It is predominantly a childhood vasculitis, rarely reported in adults. Studies have shown that several different genetic factors such as genes involved in inflammatory system and renin-angiotensin system (RAS) are important in the pathogenesis of Henoch-Schönlein purpura. OBJECTIVES: The purpose of this study was to evaluate the independent effect of 3 gene polymorphisms including CCL2-2518 C/T, VEGF-634G/C and ACE(I/D) with HSP disease and their possible joint interactions in developing the disease. MATERIAL AND METHODS: In this case-control study 47 HSP cases and 74 unrelated healthy controls were enrolled for evaluation. All individuals were genotyped for CCL2-2518C/T, VEGF-634G/C and ACE(I/D) gene polymorphisms. The possible association of these polymorphisms with susceptibility to develop HSP disease independently and in different joint combinations was evaluated. RESULTS: The frequencies of TT genotype and T allele of CCL2-2518C/T gene polymorphism and CC genotype and C allele of VEGF-634G/C gene polymorphism were significantly high in HSP children (p-values = 0.005 and = 0.007 respectively). Interestingly, studying the joint interaction of these 2 genotypes (CC genotype of VEGF G-634C and TT genotype of CCL2 C-2518T) in this cohort showed a more significant effect in the development of the disease (p < 0.000, OR = 6.009). The frequency of TT genotype of CCL2 gene when combined with II genotype of ACE gene in HSP children was significantly higher (p < 0.000, OR = 4.213). CONCLUSIONS: The results of this pilot study provide evidence of the possible gene-gene interaction effects of CCL2, VEGF and ACE genes in developing HSP disease.

Endothelial cells' biophysical, biochemical, and chromosomal aberrancies in high-glucose condition within the diabetic range.

To date, many studies have been conducted to find out the underlying mechanisms of hyperglycemia-induced complications in diabetes mellitus, attributed to the cellular pathologies of different cells-especially endothelial cells. However, there are still many ambiguities and unresolved issues to be clarified. Here, we investigated the alteration in biophysical and biochemical properties in human umbilical vein endothelial cells exposed to a high-glucose concentration (30mM), comparable to glucose content in type 2 diabetes mellitus, over a course of 120 hours. In addition to a reduction in the rate of cell viability and induction of oxidative stress orchestrated by the high-glucose condition, the dynamic of the fatty acid profile-including polyunsaturated, monounsaturated, and saturated fatty acids-was also altered in favor of saturated fatty acids. Genetic imbalances were also detected at chromosomal level in the cells exposed to the abnormal concentration of glucose after 120 hours. Moreover, the number of tip cells (CD31+ /CD34+ ) and in vitro tubulogenesis capability negatively diminished in comparison to parallel control groups. We found that diabetic hyperglycemia was associated with a decrease in the cell-cell tight junction and upregulation in vascular endothelial cadherin and zonula occludens (ZO)-1 molecules after 72 and 120 hours of exposure to the abnormal glucose concentration, which resulted in a profound reduction in transendothelial electrical resistance. The surface plasmon resonance analysis of the human umbilical vein endothelial cells immobilized on gold-coated sensor chips confirmed the loosening of the cell to cell intercellular junction as well as stable attachment of each cell to the basal surface. Our findings highlighted the disturbing effects of a diabetic hyperglycemia on either biochemical or biophysical properties of endothelial cells.

The impact of different extracellular matrices on melatonin effect in proliferation and stemness properties of ovarian cancer cells.

AIM: Endogenous melatonin has numerous physiological roles on modulating the function of different organs. Recent studies revealed oncostatic and protective effects of this molecule on tumor development. In this study, we examined the impact of melatonin and key underlying mechanisms on stemness, morphology, invasiveness and viability of SKOV3 ovarian cancer cells in different types of extracellular matrix. METHODS: Cell viability was evaluated by MTT Assay. Colony-forming assay was performed by seeding 4×103 cells on different matrices in six well-plate. The percentage of cancer stem like cells was determined by flow cytometric assay after applying antibodies against stemness markers, CD133 and CD44. Different types of extracellular matrix including fibronectin, gelatin, collagen and matrigel were applied to incubate the cells in the presence of melatonin. Downstream gene expressions including VEGF and E-cadherin were determined by Real-time PCR. RESULTS: Melatonin (0.1mM) decreased the percentage of viable cells up to 61.79±8.2% (p<0.05). Colony formation assay revealed the significant impact of melatonin in inhibition of colony formation in these cells. The maximum effect was shown in the cells incubated with melatonin on gelatin (p<0.05). Identification of stemness markers showed that applying matrigel caused significant increase in the percentage of cancer stem like cells compared to other types of extracellular matrix (p<0.05). However melatonin slightly diminished the number of stem cell like cells in all incubated matrices. Our results from gene expression assays revealed that melatonin induced a marked increase in E-cadherin along with decrease in VEGF expression levels (p<0.05). CONCLUSION: Our results suggest that interaction between ovarian cancer cells and neighboring matrices determines the subsequent anti invasive activities of melatonin.

Effect of hydroxychloroquine on oxidative/nitrosative status and angiogenesis in endothelial cells under high glucose condition.

Introduction: Under the diabetic condition, sustained production of oxidative/nitrosative stress results in irreversible vascular injuries. A great number of diabetic pathologies, such as inefficient or aberrant neo-angiogenesis emerge following chronic hyperglycemic condition. Lack of enough data exists regarding hydroxychloroquine (HCQ) contribution on angiogenesis during diabetes mellitus. Methods: To better address whether HCQ could blunt or exacerbate oxidative status and angiogenesis under high glucose condition (HCG), human umbilical vein endothelial cells (HUVECs) were exposed to 30 µM HCQ in combination with 30 mM glucose over a course of 72 hours. Viability was measured was evaluated by MTT assay. We used Griess method and TBARS assay to monitor changes in the levels of NO and MDA followed by flow cytometric analysis of ROS using DCFDA. To show the impact of HCQ on cell motility and in vitro angiogenic properties, we exploited routine scratch test and in vitro tubulogenesis, respectively. Results: Our data showed that HCQ diminished cell viability under 5 and 30 mM glucose contents. HCQ significantly decreased the total levels of nitric oxide (NO), malondialdehyde (MDA), and reactive oxygen species (ROS) in both sets of environments. Additionally, inhibitory effects were observed on cell migration after exposure to HCQ (P < 0.001). Anti-angiogenic activity of HCQ was confirmed by the reduction of tube areas under a normal or surplus amount of glucose (P < 0.001). Conclusion: In overall, results suggest that HCQ changes the oxidative/nitrosative status of HUVECs both in 5 and 30 mM conditions. HCQ is able to reduce migration and angiogenic activity of HUVECs irrespective of the glucose content.