Project description:Diabetes promotes pro-atherogenic and pro-inflammatory phenotype of VSMC associated with vascular complications. Emerging evidence shows that miRNAs play key roles in VSMC, but their role in diabetes induced effects is unclear. We profiled novel diabetes induced miRNAs in VSMC from diabetic mice and examined their role in VSMC dysfunction. High throughput small RNA-sequencing identified 135 differentially expressed miRNAs in VSMC from type 2 diabetic db/db mice (db/dbVSMC) versus non-diabetic db/+ mice. Several of these miRNA were known to regulate VSMC functions. We further focused on miR-504, because it was highly upregulated in db/dbVSMC, but its function in VSMC is unknown. We demonstrated that miR-504 and its host gene Fgf13 were upregulated in VSMC and in vivo in aortas from db/db mice. Bioinformatics analysis predicted that miR-504 targets, including a signaling adaptor Grb10 and a transcription factor Egr2, could regulate growth factor signaling. We experimentally verified that miR-504 targets Grb10 and Egr2, and it enhanced ERK1/2 activation, proliferation and migration in VSMC. Silencing Grb10 by small interfering RNA also enhanced ERK1/2 activation, inflammatory gene expression and migration. Whereas, Egr2 silencing downregulated anti-inflammatory SOCS1 and increased inflammatory genes. Furthermore, treatment with high-glucose and palmitic acid upregulated miR-504, inhibited Grb10 and enhanced inflammatory genes in VSMC. Diabetes mis-regulates expression of several miRNAs including miR-504 involved in VSMC functions. Changes in miRNAs are sustained in non-diabetic conditions suggesting involvement of miRNA dependent mechanisms in metabolic memory implicated in vascular complications. Targeting such mechanisms could offer novel therapies strategies.
Project description:We report the application of small RNA sequencing for high-throughput profiling of small RNA under 75 bp in vascular smooth muscle cell. By a reading depth of 30M and single stranded sequencing, we generated the small RNA signature on differentiated and de-differentiated vascular smooth muscle cell induced by PDGF-BB and H3K4me2 editing. We found that PDGF-BB and H3K4me2 editing induced de-differentiation modulated miRNA profile significantly, which was demonstrated at least in part responsible for modulated vascular smooth muscle cell phenotype.
Project description:To study the impact of the organotypic assembly of vascular smooth muscle cells on their transcriptome, we cultured human umbilical artery smooth muscle cells under 2D conditions and as aggregates in hanging drops under 3D conditions. After 48 hours, RNA was isolated from both groups
Project description:We used a smooth muscle cell-specific mineralocorticoid receptor knockout mouse to generate young and aged MR-intact and SMC-MR-KO aortic miRNA to examine the effect of age on vascular miRNA alterations in the presence and absence of SMC-MR. For more information about the mouse model see: McCurley, A et al. Direct regulation of blood pressure by smooth muscle cell mineralocorticoid receptors. Nat Med. 2012 Sep;18(9):1429-33 Total miRNA was extracted from young (3-4 mo) and aged male (12mo) MR-intact and SMC-MR-KO mice to investigate aging-induced alterations in vascular miRNA expression
Project description:Systemic arterial smooth muscle cells are exposed to a broad range of oxygen concentrations under physiological conditions. Hypoxia can modulate the proliferative response of smooth muscle cells leading to speculation about its role in vasculogenesis, vascular remodelling and the pathogenesis of arterial disease. The effect of hypoxia has been inconsistent, however, with both enhanced proliferation and growth arrest reported. Nevertheless, these reports support an important effect of hypoxia on smooth muscle cell proliferation and, given its physiological and clinical relevance, this requires clarification. We posited that variation in O2 concentration, within the range that exists in vivo, may have different effects on the proliferation and survival of vascular smooth muscle cells. Experiment Overall Design: Human aortic smooth muscle cells (HASMC) were propagated to passage 6 in SMGM-2 medium reached 80% confluence, the media was changed and the cells were incubated for a further 16 hrs or 48 hrs under either normoxic or hypoxic conditions (1% and 3%O2 ).
Project description:Vascular calcification is a complex process and has been associated with aging, diabetes, chronic kidney disease (CKD). Although there have been several studies studying the role of miRNAs (miRs) in bone osteogenesis, little is known about the role of miRs in vascular calcification and their role in the pathogenesis of vascular abnormalities. Matrix vesicles (MV) are known to play an important role in initiating vascular smooth muscle cell (VSMC) calcification. In the present study, we performed miRNA microarray analysis to identify the dysregulated miRs between MV and VSMC derived from CKD rats to understand the role of post-transcriptional regulatory networks governed by these miRNAs in vascular calcification and to uncover the differential miRNA content of MV. The percentage of miRNA to total RNA was increased in MV compared to VSMC. Comparison of expression profiles of miRNA by microarray demonstrated 33 miRs to be differentially expressed with the majority (~ 57%) of them down-regulated. Target genes controlled by differentially expressed miRNAs were identified utilizing two different complementary computational approaches Miranda and Targetscan to understand the functions and pathways that may be affected due to the production of MV from calcifying VSMC thereby contributing to the regulation of genes by miRs. We found several processes including vascular smooth muscle contraction, response to hypoxia and regulation of muscle cell differentiation to be enriched. Signaling pathways identified included MAP-kinase and wnt signaling that have previously been shown to be important in vascular calcification. In conclusion, our results demonstrate that miRs are concentrated in MV from calcifying VSMC, and that important functions and pathways are affected by the miRs dysregulation between calcifying VSMC and the MV they produce. This suggests that miRs may play a very important regulatory role in vascular calcification in CKD by controlling an extensive network of post-transcriptional targets. Compare miRNA from matrix vesicles to miRNA from vascular smooth muscle cells that gave rise to the matrix vesicles from 3 sets of MV and VSMC derived from 3 normal and 3 CKD rats
Project description:The full length of LncVSM transfected into Vascular Smooth Muscle cells to down-regulation for screening differential expression prolifes of LncVSM effecting.The empty vector transfected Vascular Smooth Muscle cells as controls. Eight Samples analyzed.
Project description:We used a smooth muscle cell-specific mineralocorticoid receptor knockout mouse to generate young and aged MR-intact and SMC-MR-KO aortic miRNA to examine the effect of age on vascular miRNA alterations in the presence and absence of SMC-MR. For more information about the mouse model see: McCurley, A et al. Direct regulation of blood pressure by smooth muscle cell mineralocorticoid receptors. Nat Med. 2012 Sep;18(9):1429-33
Project description:The full length of LncVSM transfected into Vascular Smooth Muscle cells to down-regulation for screening differential expression prolifes of LncVSM effecting.The empty vector transfected Vascular Smooth Muscle cells as controls.