Project description:Atherosclerosis is causally related to disturbed flow through low and oscillatory shear stress. In order to study the miR expression profile in atherosclerotic plaques induced by disturbed flow, partial ligation of the carotid artery was performed. This procedure results acutely in severly reduced blood flow and in stenotic lesion formation within 6 weeks in apoe-/- mice on a high fat diet. We compared the miR expression profile in partially ligated left carotid arteries with the untreated right carotid artery to identify miRs which are involved in plaque formation through flow disturbances. The left carotid arteries of 6 female apoe-/- mice (6-8 weeks) were partially ligated (i.e. the external and internal carotid artery as well as the occipital artery were occluded; blood flow out of the common carotid artery occurs mainly through the superior thyroid artery). Following partial ligation the animals were fed a high fat diet for 6 weeks. Total RNA was isolated from partially ligated left carotid arteries and untreated right carotid arteries (control). MiRs expression profile of the partially ligated carotid arteries were compared with the control group. Biological replicates: 6 per group. One replicate per array.
Project description:We tried to identify miRs that are differentially expresssed during atherogenesis. Aortic miRs expression profile in female apoe-/- mice after 3 and 10 months of a high fat diet were compared with female apoe-/- mice on normal diet. 4 Female apoe-/- mice (6-8 weeks) were fed on high fat diet for 3 months. 3 female apoe-/- mice (6-8 weeks) were fed on high fat diet for 10 months. 4 female apoe-/- mice (6-8 weeks) on normal diet served as controls. Total RNA was isolated from whole aortic tissue. RNA samples with RIN>8 were used for array. The aortic miRs expression profile after 3 months of a high fat diet was compared with the control group. Biological replicates: 4 per group. One replicate per array.
Project description:The global change of the miR expression profile during atherosclerosis is due to the infiltration of different types of leukocytes into the arterail vessel wall in addition to disease-specific regulation in vascular cells. Monocyte-derived macrophage accumulation in the subintimal region is critical in the formation of atherosclerotic plaques. It is currently unknown which miRs are involved in the atherogenic macrophage response. The comparison of the miR expression profile in LPS/Interferon-gamma activated mouse macrophages with the miR expression in the normal aortic vessel wall was performed to detect macrophage-enriched miRs. This screening may help to identify macrophage-enriched miRs in atherosclerotic vessels that may play a role in the macrophage function during atherogenesis. Bone marrow cells were harvested from femura of 6-8 week old female C57BL/6 mice, re-suspended in DMEM-F12/10% FCS/10% L929-conditioned medium, and cultured for 7 days to differentiate into primary macrophages. F4/80 and CD11b expression was determined by flow cytometry to confirm the macrophage phenotype. Macrophages were stimulated with LPS (100ng/ml, 14 hours) and INF-g (10ng/ml, 6 hours) and the M1 polarization was verified by quantification of mannose receptor C type 1 (MRC1), arginase II (ArgII), inducible nitric oxide synthase (iNOS), and arginase I (ArgI) by qRT-PCR. Total RNA (M1-type macrophages and aorta tissue) was isolated using mirVana microRNA Isolation Kit.
Project description:Aberrantly expressed miRNAs contribute to developmental abnormalities and diseases such as cancer, diabetes, and cardiovascular disorders, by hybridizing to specific mRNA targets and repressing their translation into proteins. Although miRNA expression signature is characterized in the process of neointimal thickening during proliferative vascular diseases such as atherosclerosis, so far global miRNA expression profiling in the different stages of atherosclerosis is completely unknown. We explored stage-specific microRNA signatures in the progress of atherosclerosis in hyperlipidemia mouse model, which may help to identify the critical miRNAs contributing atherosclerotic development and stabilization. Female apoe-/- mice (6-8 weeks, Jackson Laboratory) were fed a high fat diet (HFD, 21% crude fat, 0.15% cholesterol and 19.5% casein, Altromin, Germany) for 3 or 10 months (N=3-4). Serial sections (20 µm thick) of aortic roots and carotid arteries from these mice were mounted on membrane mounted metal frame slides (MMI), deparaffinized under RNase-free conditions, and completely dried. Laser capture microdissection (LCM) was performed with a laser microdissection system (MMI cellcut plus, Molecular Machines and Industries, Switzerland) assembled onto an inverted microscope (Olympus IX71). Plaque tissue or morphologically normal vessel wall of at least 40 sections per mouse were collected. RNA was isolated using RecoverAll total nucleic acid isolation kit (Applied Biosystems) according to the manufacturer’s instructions.
Project description:Aberrantly expressed miRNAs contribute to developmental abnormalities and diseases such as cancer, diabetes, and cardiovascular disorders, by hybridizing to specific mRNA targets and repressing their translation into proteins. Although miRNA expression signature is characterized in the process of neointimal thickening during proliferative vascular diseases such as atherosclerosis, so far global miRNA expression profiling in the different stages of atherosclerosis is completely unknown. We explored stage-specific microRNA signatures in the progress of atherosclerosis in hyperlipidemia mouse model, which may help to identify the critical miRNAs contributing atherosclerotic development and stabilization. Female apoe-/- mice (6-8 weeks, Jackson Laboratory) were fed a high fat diet (HFD, 21% crude fat, 0.15% cholesterol and 19.5% casein, Altromin, Germany) for 3 or 10 months (N=3-4). Serial sections (20 µm thick) of aortic roots and carotid arteries from these mice were mounted on membrane mounted metal frame slides (MMI), deparaffinized under RNase-free conditions, and completely dried. Laser capture microdissection (LCM) was performed with a laser microdissection system (MMI cellcut plus, Molecular Machines and Industries, Switzerland) assembled onto an inverted microscope (Olympus IX71). Plaque tissue or morphologically normal vessel wall of at least 40 sections per mouse were collected. RNA was isolated using RecoverAll total nucleic acid isolation kit (Applied Biosystems) according to the manufacturer’s instructions.
Project description:Aberrantly expressed miRNAs contribute to developmental abnormalities and diseases such as cancer, diabetes, and cardiovascular disorders, by hybridizing to specific mRNA targets and repressing their translation into proteins. Although miRNA expression signature is characterized in the process of neointimal thickening during proliferative vascular diseases such as atherosclerosis, so far global miRNA expression profiling in the different stages of atherosclerosis is completely unknown. We explored stage-specific microRNA signatures in the progress of atherosclerosis in hyperlipidemia mouse model, which may help to identify the critical miRNAs contributing atherosclerotic development and stabilization. Female apoe-/- mice (6-8 weeks, Jackson Laboratory) were fed a high fat diet (HFD, 21% crude fat, 0.15% cholesterol and 19.5% casein, Altromin, Germany) for 3 or 10 months (N=3-4). Serial sections (20 µm thick) of aortic roots and carotid arteries from these mice were mounted on membrane mounted metal frame slides (MMI), deparaffinized under RNase-free conditions, and completely dried. Laser capture microdissection (LCM) was performed with a laser microdissection system (MMI cellcut plus, Molecular Machines and Industries, Switzerland) assembled onto an inverted microscope (Olympus IX71). Plaque tissue or morphologically normal vessel wall of at least 40 sections per mouse were collected. RNA was isolated using RecoverAll total nucleic acid isolation kit (Applied Biosystems) according to the manufacturer’s instructions.
Project description:Aberrantly expressed miRNAs contribute to developmental abnormalities and diseases such as cancer, diabetes, and cardiovascular disorders, by hybridizing to specific mRNA targets and repressing their translation into proteins. Although miRNA expression signature is characterized in the process of neointimal thickening during proliferative vascular diseases such as atherosclerosis, so far global miRNA expression profiling in the different stages of atherosclerosis is completely unknown. We explored stage-specific microRNA signatures in the progress of atherosclerosis in hyperlipidemia mouse model, which may help to identify the critical miRNAs contributing atherosclerotic development and stabilization. Female apoe-/- mice (6-8 weeks, Jackson Laboratory) were fed a high fat diet (HFD, 21% crude fat, 0.15% cholesterol and 19.5% casein, Altromin, Germany) for 3 or 10 months (N=3-4). Serial sections (20 µm thick) of aortic roots and carotid arteries from these mice were mounted on membrane mounted metal frame slides (MMI), deparaffinized under RNase-free conditions, and completely dried. Laser capture microdissection (LCM) was performed with a laser microdissection system (MMI cellcut plus, Molecular Machines and Industries, Switzerland) assembled onto an inverted microscope (Olympus IX71). Plaque tissue or morphologically normal vessel wall of at least 40 sections per mouse were collected. RNA was isolated using RecoverAll total nucleic acid isolation kit (Applied Biosystems) according to the manufacturer’s instructions.
Project description:The global change of the miR expression profile during atherosclerosis is due to the infiltration of different types of leukocytes into the arterial vessel wall in addition to disease-specific regulation in vascular cells. Monocyte-derived macrophage accumulation in the subintimal region is critical in the formation of atherosclerotic plaques. It is currently unknown which miRs are involved in the atherogenic macrophage response. The comparison of the miR expression profile in LPS/Interferon-gamma activated mouse macrophages with the miR expression in the unstimulated mouse macrophages was performed to detect M1-type macrophage-enriched miRs. This screening combined with our miR profiling in atherosclerotic vessels may help to identify M1-type macrophage-enriched miRs in atherosclerotic vessels that may play a role in the macrophage function during atherogenesis. Bone marrow cells were harvested from femura of 6- to 8-week-old female C57BL/6 mice, re-suspended in DMEM-F12/10% FCS/10% L929-conditioned medium, and cultured for 7 days to differentiate into primary macrophages. F4/80 and CD11b expression was determined by flow cytometry to confirm the macrophage phenotype. Macrophages were stimulated with LPS (100ng/ml, 14 hours) and INF-g (10ng/ml, 6 hours), and the M1 polarization was verified by quantification of mannose receptor C type 1 (MRC1), arginase II (ArgII), inducible nitric oxide synthase (iNOS), and arginase I (ArgI) by qRT-PCR. Total RNA (M1-type and unstimulated (MФ) macrophages) was isolated using the mirVana microRNA Isolation Kit.
Project description:We performed miRNA and mRNA profiling over a 7-point time course, encompassing all recognized stages of lung development and explore dynamically regulated miRNAs and potential miRNA-mRNA interaction networks specific to mouse lung development replicated time course of mouse lung development in 7 time points