Klf4 has an unexpected protective role in perivascular cells within the microvasculature.
ABSTRACT: Recent smooth muscle cell (SMC) lineage-tracing studies have revealed that SMCs undergo remarkable changes in phenotype during development of atherosclerosis. Of major interest, we demonstrated that Kruppel-like factor 4 (KLF4) in SMCs is detrimental for overall lesion pathogenesis, in that SMC-specific conditional knockout of the KLF4 gene ( Klf4) resulted in smaller, more-stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage- and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. We tested the hypothesis that KLF4-dependent SMC transitions play an important role in the tissue injury-repair process. Using SMC-specific lineage-tracing mice positive and negative for simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart after ischemia-reperfusion injury (IRI) express KLF4 and transition to a KLF4-dependent macrophage-like state and a KLF4-independent myofibroblast-like state. Moreover, heart failure after IRI was exacerbated in SMC Klf4 knockout mice. Surprisingly, we observed a significant cardiac dilation in SMC Klf4 knockout mice before IRI as well as a reduction in peripheral resistance. KLF4 chromatin immunoprecipitation-sequencing analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways, including PDGF and FGF. Moreover, microvascular tissue beds in SMC Klf4 knockout mice had gaps in lineage-traced SMC coverage along the resistance arteries and exhibited increased permeability. Together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs: it is required for normal SMC function and coverage of resistance arteries. NEW & NOTEWORTHY We report novel evidence that the Kruppel-like factor 4 gene ( Klf4) has a critical maintenance role within microvascular smooth muscle cells (SMCs). SMC-specific Klf4 knockout at baseline resulted in a loss of lineage-traced SMC coverage of resistance arteries, dilation of resistance arteries, increased blood flow, and cardiac dilation.
Project description:Previous studies investigating the role of smooth muscle cells (SMCs) and macrophages in the pathogenesis of atherosclerosis have provided controversial results owing to the use of unreliable methods for clearly identifying each of these cell types. Here, using Myh11-CreER(T2) ROSA floxed STOP eYFP Apoe(-/-) mice to perform SMC lineage tracing, we find that traditional methods for detecting SMCs based on immunostaining for SMC markers fail to detect >80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages, including macrophages and mesenchymal stem cells (MSCs). SMC-specific conditional knockout of Krüppel-like factor 4 (Klf4) resulted in reduced numbers of SMC-derived MSC- and macrophage-like cells, a marked reduction in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness as compared to wild-type controls. On the basis of in vivo KLF4 chromatin immunoprecipitation-sequencing (ChIP-seq) analyses and studies of cholesterol-treated cultured SMCs, we identified >800 KLF4 target genes, including many that regulate pro-inflammatory responses of SMCs. Our findings indicate that the contribution of SMCs to atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.
Project description:RATIONALE:Elastin is an important ECM (extracellular matrix) protein in large and small arteries. Vascular smooth muscle cells (SMCs) produce the layered elastic laminae found in elastic arteries but synthesize little elastin in muscular arteries. However, muscular arteries have a well-defined internal elastic lamina (IEL) that separates endothelial cells (ECs) from SMCs. The extent to which ECs contribute elastin to the IEL is unknown. OBJECTIVE:To use targeted elastin (Eln) deletion in mice to explore the relative contributions of SMCs and ECs to elastic laminae formation in different arteries. METHODS AND RESULTS:We used SMC- and EC-specific Cre recombinase transgenes with a novel floxed Eln allele to focus gene inactivation in mice. Inactivation of Eln in SMCs using Sm22aCre resulted in depletion of elastic laminae in the arterial wall with the exception of the IEL and SMC clusters in the outer media near the adventitia. Inactivation of elastin in ECs using Tie2Cre or Cdh5Cre resulted in normal medial elastin and a typical IEL in elastic arteries. In contrast, the IEL was absent or severely disrupted in muscular arteries. Interruptions in the IEL resulted in neointimal formation in the ascending aorta but not in muscular arteries. CONCLUSIONS:Combined with lineage-specific fate mapping systems, our knockout results document an unexpected heterogeneity in vascular cells that produce the elastic laminae. SMCs and ECs can independently form an IEL in most elastic arteries, whereas ECs are the major source of elastin for the IEL in muscular and resistance arteries. Neointimal formation at IEL disruptions in the ascending aorta confirms that the IEL is a critical physical barrier between SMCs and ECs in the large elastic arteries. Our studies provide new information about how SMCs and ECs contribute elastin to the arterial wall and how local elastic laminae defects may contribute to cardiovascular disease.
Project description:<h4>Background</h4>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 risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC.<h4>Methods</h4>We combined RNA-sequencing, chromatin immunoprecipitation followed by sequencing, assay for transposase-accessible chromatin using sequencing, and in vitro assays in human coronary artery SMCs, with single-cell RNA-sequencing, histology, and RNAscope in an SMC-specific lineage-tracing <i>Ahr</i> knockout mouse model of atherosclerosis to better understand the role of <i>AHR</i> in vascular disease.<h4>Results</h4>Genomic studies coupled with functional assays in cultured human coronary artery SMCs revealed that <i>AHR</i> modulates the human coronary artery SMC phenotype and suppresses ossification in these cells. Lineage-tracing and activity-tracing studies in the mouse aortic sinus showed that the <i>Ahr</i> pathway is active in modulated SMCs in the atherosclerotic lesion cap. Furthermore, single-cell RNA-sequencing studies of the SMC-specific <i>Ahr</i> knockout mice showed a significant increase in the proportion of modulated SMCs expressing chondrocyte markers such as <i>Col2a1</i> and <i>Alpl</i>, which localized to the lesion neointima. These cells, which we term "chondromyocytes," 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 SMCs in the lesion cap, and increased alkaline phosphatase activity in lesions in the <i>Ahr</i> knockout in comparison with wild-type mice. We propose that <i>AHR</i> is likely protective based on these data and inference from human genetic analyses.<h4>Conclusions</h4>Overall, we conclude that <i>AHR</i> promotes the maintenance of lesion cap integrity and diminishes the disease-related SMC-to-chondromyocyte transition in atherosclerotic tissues.
Project description:The role of microRNA-1 (miR-1) has been studied in cardiac and skeletal muscle differentiation. However, it remains unexplored in vascular smooth muscle cells (SMCs) differentiation. The aim of this study was to uncover novel targets of and shed light on the function of miR-1 in the context of embryonic stem cell (ESC) differentiation of SMCs in vitro. miR-1 expression is steadily increased during differentiation of mouse ESC to SMCs. Loss-of-function approaches using miR-1 inhibitors uncovered that miR-1 is required for SMC lineage differentiation in ESC-derived SMC cultures, as evidenced by downregulation of SMC-specific markers and decrease of derived SMC population. In addition, bioinformatics analysis unveiled a miR-1 binding site on the Kruppel-like factor 4 (KLF4) 3' untranslated region (3'UTR), in a region that is highly conserved across species. Consistently, miR-1 mimic reduced KLF4 3'UTR luciferase activity, which can be rescued by mutating the miR-1 binding site on the KLF4 3'UTR in the reporter construct. Additionally, repression of the miR-1 expression by miR-1 inhibitor can reverse KLF4 downregulation during ESC-SMC differentiation, which subsequently inhibits SMC differentiation. We conclude that miR-1 plays a critical role in the determination of SMC fate during retinoid acid-induced ESC/SMC differentiation, which may indicate that miR-1 has a role to promote SMC differentiation.
Project description:Resident vascular adventitial progenitor cells express the stem cell marker, Sca1 (AdvSca1 cells). Using smooth muscle cell (SMC) lineage tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) that originate from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. The adventitial microenvironment and induction of the transcription factor, Klf4 is critical to reprogramming; however, the mechanism of Klf4 induction is unknown. We aimed to define the signaling pathways involved in SMC reprogramming and the fate of AdvSca1-SM cells in response to vascular injury. Flow sorting was used to isolate YFP+Sca1- SMCs, YFP+Sca1+ AdvSca1-SM cells, and YFP-Sca1+ non-SMC-derived AdvSca1 cells from aortae (AO) and carotid arteries (CA) of SMC YFP reporter mice. RNA-seq analysis and unbiased hierarchical clustering revealed that genes related to hedgehog/Wnt/-catenin signaling are significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in SMC reprogramming. CA injury was induced by ligating the left common CA of SMC YFP reporter mice. In response to injury, AdvSca1-SM cells downregulate genes in the hedgehog/Wnt/-catenin signaling pathway, as well as stemness-related genes, and adopt a myofibroblast-like phenotype. To selectively fate-map AdvSca1-SM cells, we took advantage of the selective expression of Gli1 by AdvSca1-SM cells and generated Gli1-CreERT2-Rosa26-YFP reporter mice. Immunofluorescent (IF) staining show that YFP was expressed exclusively in a subpopulation of adventitial Sca1-positive cells. RNA-seq confirmed YFP+Sca1+ cell population in SMC and Gli1 lineage tracing models exhibit a highly similar gene expression profile, supporting this model to faithfully track AdvSca1-SM cells. CA injury induced proliferation and differentiation of AdvSca1-SM cells to myofibroblasts rather than macrophages, as indicated by IF stainings and flow cytometry. Surprisingly, AdvSca1-SM cells selectively contributed to adventitial remodeling and fibrosis, but little neointima formation. AdvSca1-SM cell specific genetic knockout of Klf4 induced spontaneous differentiation and expansion of AdvSca1-SM cells and increased perivascular fibrosis. Overall design: RNA gene expression profile from mouse vascular smooth muscle cells (SMCs, 3 independent replicates), SMC-derived vascular AdvSca1 cells (AdvSca1-SM, 2 independent replicates) , and non-SMC-derived AdvSca1 cells (AdvSca1-MA, 2 independent replicates) sorted from aortae and carotid arteries of SMC lineage tracing mouse models, and AdvSca1-SM cells (3 independent replicates) from the aortae and carotid arteries of Gli1 lineage tracing mouse model. RNA gene expression profile of AdvSca1-SM cells sorted from injured (4 independent replicates) and uninjured (4 independent replicates) carotid arteries of SMC lineage tracing mouse model.
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:Elastic fibers are critical for the mechanical function of the large arteries. Mechanical effects of elastic fiber protein deficiency have been investigated in whole arteries, but not in isolated smooth muscle cells (SMCs). The elastic moduli of SMCs from elastin (Eln-/-) and fibulin-4 (Fbln4-/-) knockout mice were measured using atomic force microscopy. Compared to control SMCs, the modulus of Eln-/- SMCs is reduced by 40%, but is unchanged in Fbln4-/- SMCs. The Eln-/- SMC modulus is rescued by soluble or ? elastin treatment. Altered gene expression, specifically of calponin, suggests that SMC phenotypic modulation may be responsible for the modulus changes.
Project description:<h4>Background</h4>Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a 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.<h4>Methods</h4>We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis.<h4>Results</h4>We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11<sup>-</sup>, Lgals3<sup>+</sup> population with a chondrocyte-like gene signature that was markedly reduced with SMC-<i>Klf4</i> knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. 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-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives 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.<h4>Conclusions</h4>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<sup>+</sup> osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.
Project description:Smooth muscle cells (SMCs) express a unique set of microRNAs (miRNAs) which regulate and maintain the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal (GI) SMCs in a transgenic animal model. We generated SMC-specific Dicer null animals that express the reporter, green fluorescence protein, in a SMC-specific manner. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs: the mutant mice developed severe dilation of the intestinal tract associated with the thinning and destruction of the smooth muscle (SM) layers; contractile motility in the mutant intestine was dramatically decreased; and SM contractile genes and transcriptional regulators were extensively down-regulated in the mutant SMCs. Profiling and bioinformatic analyses showed that SMC phenotype is regulated by a complex network of positive and negative feedback by SMC miRNAs, serum response factor (SRF), and other transcriptional factors. Taken together, our data suggest that SMC miRNAs are required for the development and survival of SMCs in the GI tract.
Project description:Phenotypic switching of smooth muscle cells (SMCs) plays a key role in vascular proliferative diseases. We previously showed that Krüppel-like factor 4 (Klf4) suppressed SMC differentiation markers in cultured SMCs. Here, we derive mice deficient for Klf4 by conditional gene ablation and analyze their vascular phenotype following carotid injury. Klf4 expression was rapidly induced in SMCs of control mice after vascular injury but not in Klf4-deficient mice. Injury-induced repression of SMC differentiation markers was transiently delayed in Klf4-deficient mice. Klf4 mutant mice exhibited enhanced neointimal formation in response to vascular injury caused by increased cellular proliferation in the media but not an altered apoptotic rate. Consistent with these findings, cultured SMCs overexpressing Klf4 showed reduced cellular proliferation, in part, through the induction of the cell cycle inhibitor, p21(WAF1/Cip1) via increased binding of Klf4 and p53 to the p21(WAF1/Cip1) promoter/enhancer. In vivo chromatin immunoprecipitation assays also showed increased Klf4 binding to the promoter/enhancer regions of the p21(WAF1/Cip1) gene and SMC differentiation marker genes following vascular injury. Taken together, we have demonstrated that Klf4 plays a critical role in regulating expression of SMC differentiation markers and proliferation of SMCs in vivo in response to vascular injury.