Project description:Coronary artery disease is the leading cause of mortality in women. Abnormal growth of smooth muscle cells and endothelial damage contributes to the thickening of the blood vessels and vascular occlusion. Estradiol prevents vascular remodleing by promoting EC growth and inhibiting SMC growth. Recent studies suggest that circulating (bone marrow derived) progenitor cells also contribute to the vascular remodeling process. Whether estradiol could, in part, induce its protective actions by inhibiting P-SMC growth remains unknown. In this study, progenitor smooth muscle cells (P-SMCs) and endothelial (P-ECs) grown from CD34+ progenitor cells (PC) magnetically separated from human cord blood derived mononuclear cells. P-SMC and P-EC cell colonies were grown and characterized for SMC and EC markers. Cultured cells were further used to assess the effects of 17β-estradiol (E2; 10nM) on their growth and gene expression. Briefly, P-SMCs or P-ECs were treated with or without 10 nM E2 for 0, 4 or 30 hours in DMEM-F12 supplemented with 2.5% FCS (steroid-free; SF). Subsequently, total RNA was extracted using RNeasy Mini Kit (QIAGEN) processed for gene analysis. Treatment of P-SMCs with E2 inhibited 2.5% SF-serum -induced growth (DNA synthesis and cell number), whereas, E2 induced cell growth in P-ECs. The growth effects of E2 in P-SMCs and P-ECs were mimicked by ER-α agonist and blocked by ER-α antagonist. The differential effects of E2 on P-SMC and P-EC growth were also reflected in ERK1/2 and Akt phosphorylation and in positive and negative regulators of cell cycle. Gene analysis revealed that P-SMCs expressed SMC alpha actin (ACTA2) where as P-ECs expressed CD34, FLT1 and vWF, confirming the differentiation of progenitor cells to SMCs and ECs. Treatment with E2 for 30 hours regulated 1791 in P-SMCs and 220 in P-ECs, with 36 that were common. Volcano analysis showed that treatment with E2 for 30 hours differentially expressed multiple genes, importantly, estradiol significantly upregulated antimitogenic pathways and downregulated proproliferative pathways in P-SMC and P-ECs, respectively. Contrasting effects of E2 were also observed in multiple ingenuity canonical pathways that were up or downregulated in P-SMCs and P-ECs. Findings from both growth and gene expression studies provide evidence that etradiol may induce its vascular protective actions by inhibiting P-SMC activity and improving P-EC activity. Interestingly, in perpheral blood estradiol decreasd the number of c-kit+ cells and increased CD34+ cells. Since these cells evolve into SMCs and ECs respectively, our findings provide evidence that estradiol may induce its anti-occlusive actions by differentially regulating P-SMC and P-EC activity and numbers.

Project description:Tissue Factor Pathway Inhibitor-2 is Induced by Fluid Shear Stress in Vascular Smooth Muscle Cells and Affects Cell Proliferation and Survival Introduction: Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. Methods: Laminar FSS of arterial level (14 dynes/cm2) was applied to SMC cultures for 24 h in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology and the results further verified by real time (RT) PCR and immunoblotting. Protein expression was also studied in the rat carotid artery after balloon-injury and DNA synthesis and apoptosis was examined in SMCs in vitro. Results: Microarrays identified tissue-pathway inhibitor-2 (TFPI-2) as the most differentially expressed gene by FSS in cultured SMCs. The regulatory effect of FSS on the expression of TFPI-2 was confirmed by RT-PCR and immunobloting demonstrating a more than 400-fold increase in TFPI-2 expression in SMCs exposed to FSS compared to static controls and a consistent upregulation at the protein level. Functionally, SMC proliferation was decreased by FSS and recombinant TFPI-2 was found to inhibit SMC proliferation and induce SMC apoptosis as indicated by activation of caspase-3. In vivo, TFPI-2 expression was found to be up-regulated 5, 10 and 20 h after rat carotid balloon-injury and immunohistochemistry demonstrated TFPI-2 protein in luminal SMCs exposed to FSS in rat carotid intimal hyperplasia 10 days after balloon-injury. Conclusion: FSS strongly influence gene expression in cultured SMCs and induce TFPI-2 expression, which is also expressed after rat carotid balloon injury in luminal SMCs exposed to FSS. Functionally, TFPI-2 may play an important role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 control SMC function. 3 x 2 samples from a paired fluid shear stress experiment on smooth muscle cells.

Project description:Tissue Factor Pathway Inhibitor-2 is Induced by Fluid Shear Stress in Vascular Smooth Muscle Cells and Affects Cell Proliferation and Survival Introduction: Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. Methods: Laminar FSS of arterial level (14 dynes/cm2) was applied to SMC cultures for 24 h in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology and the results further verified by real time (RT) PCR and immunoblotting. Protein expression was also studied in the rat carotid artery after balloon-injury and DNA synthesis and apoptosis was examined in SMCs in vitro. Results: Microarrays identified tissue-pathway inhibitor-2 (TFPI-2) as the most differentially expressed gene by FSS in cultured SMCs. The regulatory effect of FSS on the expression of TFPI-2 was confirmed by RT-PCR and immunobloting demonstrating a more than 400-fold increase in TFPI-2 expression in SMCs exposed to FSS compared to static controls and a consistent upregulation at the protein level. Functionally, SMC proliferation was decreased by FSS and recombinant TFPI-2 was found to inhibit SMC proliferation and induce SMC apoptosis as indicated by activation of caspase-3. In vivo, TFPI-2 expression was found to be up-regulated 5, 10 and 20 h after rat carotid balloon-injury and immunohistochemistry demonstrated TFPI-2 protein in luminal SMCs exposed to FSS in rat carotid intimal hyperplasia 10 days after balloon-injury. Conclusion: FSS strongly influence gene expression in cultured SMCs and induce TFPI-2 expression, which is also expressed after rat carotid balloon injury in luminal SMCs exposed to FSS. Functionally, TFPI-2 may play an important role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 control SMC function.

Project description:Smooth muscle cells (SMCs) display dynamic plasticity by changing phenotype under various pathophysiological conditions. Uncovering how SMCs regulate their phenotype is a key to understanding the molecular mechanisms of a number of gastrointestinal diseases. MicroRNAs (miRNAs), generated in cells by Dicer, have been identified as regulators of the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal SMCs in a transgenic animal model. We generated SMC-specific Dicer (smDicer) null animals that express the reporter, green fluorescence protein (eGFP), in a SMC-specific manner. eGFP labeled SMCs were used for morphological, cytometric and genetic studies of smDicer null mutant and wild type control mice. The structure of the bowel wall was examined by confocal microscopy, and function was evaluated by recording spontaneous and evoked contractions. SMCs were purified with fluorescence-activated cell sorting and SM specific gene expression studies were performed with genechip arrays, PCR and quantitative PCR. Bioinformatic analyses were used to characterize the interaction between miRNAs and target genes. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs. Profiling and bioinformatic analyses showed SMC phenotype is regulated by a complex network of positive and negative feedback by SM miRNAs, serum response factor (SRF), and other transcriptional factors. SM miRNAs play an important role in growth, development and survival of SMCs. Phenotypic changes of SMCs may be regulated through multiple pathways in an interaction network of SM miRNAs, SRF, and additional transcriptional factors. We used microarrays to identify genetic changes during the development of gastric smooth muscle cells in the smDicer null mice. Mouse mRNA microarrays were performed on GeneChipÂ® Mouse Genome 430 2.0 Arrays (Affymetrix). Total RNAs isolated from the jejunal muscularis of the smDicer knockout (KO) and the wild type (WT) control mice at the ages of ~3 weeks were used for the arrays. The cDNA synthesis, hybridization, cDNA labeling, and scanning were performed. Gene expression between the smDicer KO and the WT controls were compared.

Project description:Background: Palmdelphin (PALMD), a protein of unknown function, belongs to the family of Paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms (SNPs) in the PALMD locus, which reduce its expression, are strong risk factors for development of calcific aortic valve stenosis (CAVS) and for severity of the disease. Methods: Public database screening and immunodetection of PALMD showed dominant expression in endothelial cells (ECs). Mass spectrometry allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in siRNA silenced EC cultures, in knockout mice and in human valve samples and EC cultures from patients. RNA sequencing on siRNA silenced cells and transcript arrays on valve samples from a Swedish SNP rs7543130 patient cohort informed about gene regulatory changes. Results: ECs express the cytoplasmic PALMD-KKVI splice variant, which was shown to exist in complex with RAN GTPase activating protein1 (RANGAP1). RANGAP1 regulates the activity of the GTPase RAN and thereby, nucleocytoplasmic shuttling via Exportin1 (XPO1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, providing a mechanism for the gene regulatory changes established in PALMD-deficiency. Gene ontology analysis showed enrichment of actin and RHO GTPAse related terms in PALMD-silenced ECs. In accordance, PALMD-silenced ECs showed multiple changes in adhesive and migratory properties and cells were softer. In particular, PALMD-deficient ECs failed to form a perinuclear actin cap when exposed to flow. The perinuclear actin cap is known to provide protection against mechanical stress. Lack of the actin cap correlated with tilting of the nuclear long axis relative to the cell body, which was established in PALMD-deficient ECs, mice and patient valve samples. The loss of the actin cap may contribute to further dysregulated gene transcription eventually promoting calcific precipitations and inflammation in CAVS. Conclusions: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex changes the subcellular localization of essential nucleocytoplasmic shuttling components, thereby instigating the loss of actin-dependent nuclear resilience, which in turn promotes further deterioration of gene expression patterns predisposing to the establishment of CAVS.

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Analysis of Combined Transcriptomes Identifies Gene Modules Differentially Responding to Pathogenic Stimulation in Vascular Smooth Muscle and Endothelial Cells

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