Adhesion of monocytes and endothelial cells isolated from the human aorta suppresses by miRNA-PEI particles.

BACKGROUND: Knowledge of stenosis in coronary arteries requires an understanding of the cellular and molecular processes that occur throughout the leukocyte rolling process. In this study, the roles of miR-125a-5p and miR-495-3p were investigated on the adhesion of endothelial cells (ECs) isolated from the human aorta. METHODS: Human primary endothelial cells were obtained from the aorta of people who had died of brain death. Whole blood was used to isolate the monocytes. The miR-125 and miR-495 were predicted and transfected into ECs using Poly Ethylene Imine (PEI). The expression levels of adhesion molecules and monocyte recruitment were identified by the RT-qPCR technique and Leukocyte-Endothelial Adhesion Assay kit, respectively. RESULTS: The ICAM-1, ICAM-2 and VCAM-1 expression levels decreased significantly in the miR-495/PEI-transfected ECs (P < 0.05) while in the miR-125/PEI-transfected ECs only the ICAM-2 and ITGB-2 expression levels decreased significantly (P < 0.05) as compared to the miR-synthetic/PEI-transfected ECs. Furthermore, the monocyte adhesion was decreased in the miR-125 and miR-mix/PEI-transfected ECs as compared to the miR-synthetic/PEI-transfected ECs (P = 0.01 and P = 0.04, respectively). CONCLUSION: According to the findings, the efficient relations between miR-125 and adhesion molecules may be responsible for the inhibition of monocyte rolling.

Effect of a Peptide Construct on Differentiated Macrophage MMP-2 and MMP-9 Levels of Varicose Patients.

BACKGROUND: The Matrix Metalloproteinase (MMPs) secreted from macrophages can affect the extracellular matrix remodeling process and improve varicose veins. AIM: The aim of this study was to investigate the MMP-2 and MMP-9 gene expression and activity levels in the differentiated macrophages M2 of subjects with varicose veins, and to evaluate a peptide construct on their catalytic functions. METHODS: The macrophages were differentiated from the monocytes using M-CSF. The MMP-2 and MMP-9 gene expression and activity levels were measured by RT-qPCR and Zymography techniques, respectively. A peptide construct (ESLCG) was predicted with bioinformatics tools, and was prepared for the study of enzyme functions as compared to Batimastat. Furthermore, the docking studies were obtained for the evaluation of interactions between peptide construct, Batimastat and enzyme 3D structures. RESULTS: The results showed significant increases in MMP2 and MMP9 gene expression levels (P<0.001 and P<0.004, respectively) and gelatinolytic activities (P<0.001 and P<0.0001, respectively) in the macrophages. In agreement with the inhibitory effects of Batimastat, the peptide construct inhibited the MMP-2 and MMP-9 gelatinolytic activities up to 6.8 and 6.5 folds in the concentration of 150 µM. The docking analyses showed that the Lys187, Arg98, Leu49, Gly189, Leu190, Met97, Tyr53 and Phe57 residues of MMP-2 and the Leu187, His190, Glu402, His401, His405 and His411 residues of MMP-9 are interacted with the atoms of Batimastat and ESLCG peptide. CONCLUSION: The ESLCG peptide may be applied as an inhibitor of MMP-2 and MMP-9 enzymes in the subjects with varicose veins.

Isolation of intimal endothelial cells from the human thoracic aorta: Study protocol.

Background: Vessel endothelial cells are extensively applied to study the mechanism of atherosclerosis. Some cellular sources including human umbilical artery endothelial cells (HUAECs) and human umbilical vein endothelial cells (HUVECs) are mostly applied in the experimental studies. We described a method for isolating the human endothelial cells from the human thoracic aorta. Methods: Normal aortic samples were prepared from subjects with brain death in Masih Daneshvari Hospital. The endothelial cells were isolated using collagenase and were evaluated by the measurement of CD31 marker. Furthermore, the digestion efficacy was studied by vessel histological analysis, and the adhesion mechanism was investigated by leukocyte endothelial adhesion assay kit. Results and Conclusion: The isolation protocol is found as a fast and simple technique with a proper cellular load to separate the endothelial cells from the human aorta.

Associations between miR-661, miR-1202, lncRNA-HOTAIR, lncRNA-GAS5 and MMP9 in differentiated M2-macrophages of patients with varicose veins.

BACKGROUND: The venous hypertension is suggested as the main cause of varicose disease. Some mediators and growth factors are known as the responsible of cellular events for the progression of venous perturbations. The aim of this study was to investigate non-coding (nc) RNA and MMP9 expression levels in macrophages differentiated from monocytes of patients with varicose veins. METHODS: The monocytes were isolated from the whole blood samples by RosetteSep kit and were differentiated to macrophages M2 using M-CSF factor. The based on ncRNA-gene network, lncRNA-GAS5, lncRNA-HOTAIR, miRNA-661, miRNA-1202, and MMP9 were selected. The gene expression levels were measured by RT-qPCR technique. RESULTS: Data showed that the MMP9 gene expression increased (P=0.003) while the GAS5, miRNA-661, and miRNA-1202 expression levels reduced significantly in the differentiated macrophages of patients (P=0.035, P=0.009, and P=0.015, respectively). Furthermore, the MMP9 gene expression levels were conversely related to the GAS5, HOTAIR, miRNA-661 and miRNA-1202 expression levels. CONCLUSIONS: The results suggested that the lncRNA-GAS5, miRNA-661, miRNA-1202 and MMP9 are involved in varicose disease.