GSE94661 - Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions - OmicsDI

Transcriptomics

Dataset Information

Smooth muscle cell-specific deletion of Col15a1 unexpectedly leads to impaired development of advanced atherosclerotic lesions

ABSTRACT: Atherosclerotic plaque rupture with subsequent embolic events is a major cause of sudden death from myocardial infarction or stroke. Although smooth muscle cells (SMC) produce and respond to collagens in vitro, there is no direct evidence in vivo that SMC are a critical source of collagens impacting lesion development or fibrous cap formation. We sought to determine how conditional SMC specific knockout of collagen type XV (COL15A1) in SMC lineage tracing mice effects advanced lesion formation. COL15A1 was chosen because: 1) we previously identified a Col15a1 sequence variant associated with age related atherosclerosis; 2) COL15A1 is a matrix organizer that enhances tissue structural integrity; and 3) siRNA mediated Col15a1 knockdown increased migration and decreased proliferation of SMC in vitro. We hypothesized that SMC derived COL15A1 is critical in advanced lesion pathogenesis and fibrous cap formation. Surprisingly, we demonstrate that SMC specific Col15a1 knockout mice fed a Western diet for 18 weeks failed to form advanced lesions. SMC Col15a1 knockout lesions have a drastic reduction in overall lesion size, cell accumulation, and the absence of a SMC and ECM- rich lesion or fibrous cap. In vivo RNA-seq analysis on lesions +/- SMC Col15a1 knockout suggests the mechanism for these effects is complex and associated with reductions in multiple pro-atherogenic inflammatory pathways in multiple cell types involved in lesion development. These results thus provide the first direct evidence that a SMC derived collagen is critical during lesion pathogenesis. Additionally, we conclude that SMC Col15a1 knockout unexpectedly inhibited rather than exacerbated lesion pathogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE94661 | GEO | 2017/09/08

SECONDARY ACCESSION(S): PRJNA371776

REPOSITORIES: GEO

Similar Datasets

Project description:Despite decades of research, our understanding of processes controlling the stability of late-stage atherosclerotic plaques remains poor. However, a prevailing hypothesis is that reducing inflammation may improve plaque stability. Indeed, the potent inflammatory cytokine, interleukin-1β (IL-1β), has been shown to be a key driver of atherosclerosis development. Importantly, the CANTOS Trial recently demonstrated that administration of an anti-IL-1β antibody to high-risk post-myocardial infarction (MI) patients reduced the incidence of recurrent nonfatal MI, but did not reduce the incidence of cardiovascular death or stroke. As such, although the CANTOS trial results providing exciting evidence that targeting inflammation can have clinical benefit for treating advanced atherosclerosis, extensive further investigation is needed to better understand the mechanisms by which IL-1β inhibition impacts established lesions. Therefore, we performed intervention studies on smooth muscle cell (SMC) lineage tracing mice with advanced atherosclerosis using anti-IL- 1 or IgG control antibodies. Surprisingly, we found no effect on lesion size but a profound shift in the composition of the fibrous cap characterized by reduced collagen and SMC content but an increase in macrophage number, which was primarily driven by opposite effects on the proliferation of these respective cell types. By generating SMC-specific and macrophage-selective Il1r1 KO Apoe -/- mice, we found that SMC-specific Il1r1 KO resulted in a ~60% reduction in lesion size and lesions that were nearly devoid of YFP + SMC whereas, myeloid-selective loss of IL1R1 had no effect on lesion size, or cell composition. This suggests that SMC are a primary cell type responding to IL-1β in atherosclerosis and that IL1 signaling in SMC is critical for their investment and retention within the fibrous cap. In addition, we found that inhibition of IL-1β promoted an expansion of the M2 macrophage population within the fibrous cap, and that these effects may be due in part to elevated levels of IL4. Taken together, results show that IL-1β can promote beneficial changes in late-stage murine atherosclerosis by promoting maintenance of a SMC/collagen-rich fibrous cap. Moreover, studies identify critical cell types and pathways that need to be considered when attempting to develop safer and more effective anti-inflammatory therapies for widespread treatment of atherosclerotic disease, including in moderate or low risk patients.

Project description:Cardiovascular disease is the leading cause of death in developed countries and there is compelling evidence that the majority of these fatalities are secondary to rupture of unstable atherosclerotic plaques. However, the mechanisms that control plaque stability are poorly understood, although it is widely believed that plaques having a decreased ratio of cells positive for smooth muscle cell (SMC) markers such as ACTA2 relative to macrophage markers are more likely to rupture. Herein we employMyh11-CreERT2 ROSA floxed STOP eYFP Apoe-/- mice to trace SMC lineage and show that traditional methods for detecting SMCs based on immunostaining for SMC markers like ACTA2 are unable to detect 82% of SMC-derived cells within advanced atherosclerotic lesions. Moreover, we show that these SMC-derived cells within advanced lesions exhibit multiple phenotypes including activation of multiple markers of macrophages, mesenchymal stem cells (MSC), and myofibroblasts (MF). Importantly, we show that SMC-derived macrophage like cells are phagocytic based on electron microscope YFP immunolabeling. In addition, results of single cell epigenetic assays developed in our lab shows that nearly 20% of macrophage-like cells within human lesions are of SMC not myeloid origin. These phenotypic transitions appear to be critical in lesion pathogenesis, in that we show that SMC-specific conditional knockout (KO) of the pluripotency factor, Krüppel-like factor 4 (KLF4), resulted in marked reductions in lesion size, increases in multiple indices of plaque stability, and reduced numbers of SMC-derived MSC- and macrophage-like cells. Finally, we show that cholesterol loading of cultured SMC is associated with activation of multiple markers of macrophages and MSC, secretion of pro-inflammatory cytokines, and increased phagocytosis, all of which are Klf4 dependent. Taken together, results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that loss of Klf4 within SMC result in major changes in SMC phenotype and function that play a major role in lesion pathogenesis. Examination of KLF4 transcription factor in an atherosclerosis setting

Project description:Aryl-hydrocarbon receptor protects against endochondral ossification of modulated smooth muscle cells in atherosclerosis Introduction: Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. Methods: We combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. Results: Genomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term chondromyocytes (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. Conclusion: Overall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

Project description:Background Rupture or erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a very limited understanding of the identity, origin, and function of many cells that make up late stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. Methods and Results Using RNAseq and ChIPseq of advanced atherosclerotic lesions from mice, we provide evidence SMC-specific Klf4- versus Oct4-knockout ApoE-/- mice showed virtually opposite genomic signatures and putative SMC Klf4 or Oct4 target genes play an important role regulating SMC phenotypic changes. Further, we conducted a comprehensive single-cell RNA-seq of advanced human carotid endarterectomy samples and compared these with scRNAseq from murine micro-dissected advanced atherosclerotic lesions with SMC and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. This analysis revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and EC-derived lesion cells. The latter included 7 distinct clusters of SMC- and EC-derived cells, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-specific conditional knockout of Klf4. To study these cells and the mechanisms of their transition, we developed an innovative dual lineage tracing mouse that can uniquely label and genetically target Myh11+ SMC that subsequently activate Lgals3. We observed that SMC that activate Lgals3 comprise up to 2/3 of all SMC in advanced plaques. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene+, ECM-remodeling, “pioneer” cell phenotype that are the first to invest within lesions and subsequently give rise to at least 3 other SMC phenotypes within advanced lesions including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Conclusions Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late stage atherosclerosis plaque pathogenesis.

Project description:Cardiovascular disease is the leading cause of death in developed countries and there is compelling evidence that the majority of these fatalities are secondary to rupture of unstable atherosclerotic plaques. However, the mechanisms that control plaque stability are poorly understood, although it is widely believed that plaques having a decreased ratio of cells positive for smooth muscle cell (SMC) markers such as ACTA2 relative to macrophage markers are more likely to rupture. Herein we employMyh11-CreERT2 ROSA floxed STOP eYFP Apoe-/- mice to trace SMC lineage and show that traditional methods for detecting SMCs based on immunostaining for SMC markers like ACTA2 are unable to detect 82% of SMC-derived cells within advanced atherosclerotic lesions. Moreover, we show that these SMC-derived cells within advanced lesions exhibit multiple phenotypes including activation of multiple markers of macrophages, mesenchymal stem cells (MSC), and myofibroblasts (MF). Importantly, we show that SMC-derived macrophage like cells are phagocytic based on electron microscope YFP immunolabeling. In addition, results of single cell epigenetic assays developed in our lab shows that nearly 20% of macrophage-like cells within human lesions are of SMC not myeloid origin. These phenotypic transitions appear to be critical in lesion pathogenesis, in that we show that SMC-specific conditional knockout (KO) of the pluripotency factor, Krüppel-like factor 4 (KLF4), resulted in marked reductions in lesion size, increases in multiple indices of plaque stability, and reduced numbers of SMC-derived MSC- and macrophage-like cells. Finally, we show that cholesterol loading of cultured SMC is associated with activation of multiple markers of macrophages and MSC, secretion of pro-inflammatory cytokines, and increased phagocytosis, all of which are Klf4 dependent. Taken together, results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that loss of Klf4 within SMC result in major changes in SMC phenotype and function that play a major role in lesion pathogenesis.

Project description:Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery” as well as disease-related cellular functions including “cellular movement,” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.