Herein we employ Myh11-CreERT2 ROSA floxed STOP eYFP Apoe−/− smooth muscle cell (SMC) lineage tracing mice to show that traditional methods for detecting SMCs based on immuno-staining 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 (Mϕs), and mesenchymal stem cells (MSCs). SMC-specific conditional knockout (KO) of Krüppel-like factor 4 (KLF4) resulted in reduced numbers of SMC-derived MSC-, and Mϕ-like cells, marked reductions in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness. Results of in vivo KLF4 ChIP-Seq analyses, and studies in cultured SMC treated with cholesterol identified > 800 KLF4 target genes including many that regulate pro-inflammatory responses of SMC. Results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.

Background: 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. Methods: 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. Results: 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 - , Lgals3 + population with a chondrocyte-like gene signature that was markedly reduced with SMC- Klf4 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. 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.

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.

Objective: Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4 -dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-Cre ERT2 eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP + cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP + ; and unintended proinflammatory consequences. Using newly generated Myh11-Dre ERT2 tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4 Δ/Δ ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1 + lymphatic endothelial cells in the epididymal AT. Conclusions: Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.

In the version of this article initially published, the labels to the left of the two micrographs in Figure 2c are reversed. Also, in Figure 4g, MKi67, used as a cell proliferation marker, is misspelled. The errors have been corrected in the HTML and PDF versions of the article.

Objective: Oxidized phospholipids (OxPL), such as the oxidized derivatives of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, have been shown to be the principal biologically active components of minimally oxidized LDL (low-density lipoprotein). The role of OxPL in cardiovascular diseases is well recognized, including activation of inflammation within vascular cells. Atherosclerotic Apoe –/– mice fed a high-fat diet develop antibodies to OxPL, and hybridoma B-cell lines producing natural anti-OxPL autoantibodies have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing antibodies can be used to prevent or reverse advanced atherosclerosis. Approach and Results: Here, using a screening against 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine/1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, we generated a novel IgM autoantibody, 10C12, from the spleens of Apoe –/– mice fed a long-term Western diet, that demonstrated potent OxPL neutralizing activity in vitro and the ability to inhibit macrophage accumulation within arteries of Apoe –/– mice fed a Western diet for 4 weeks. Of interest, 10C12 failed to inhibit atherosclerosis progression in Apoe –/– mice treated between 18 and 26 weeks of Western diet feeding likely due at least in part to high levels of endogenous anti-OxPL antibodies. However, 10C12 treatment caused a 40% decrease in lipid accumulation within aortas of secreted IgM deficient, sIgM –/– Apoe –/– , mice fed a low-fat diet, when the antibody was administrated between 32-40 weeks of age. Conclusions: Taken together, these results provide direct evidence showing that treatment with a single autoimmune anti-OxPL IgM antibody during advanced disease stages can have an atheroprotective outcome.

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Background: Prior studies have not clearly established risk of cardiovascular disease (CVD) among smokers who switch to exclusive use of electronic nicotine delivery systems (ENDS). We compared cardiovascular disease incidence in combustible-tobacco users, those who transitioned to ENDS use, and those who quit tobacco with never tobacco users. Methods: This prospective cohort study analyzes five waves of Population Assessment of Tobacco and Health (PATH) Study data, Wave 1 (2013–2014) through Wave 5 (2018–2019). Cardiovascular disease (CVD) incidence was captured over three intervals (Waves 1 to 3, Waves 2 to 4, and Waves 3 to 5). Participants were adults (40+ years old) without a history of CVD for the first two waves of any interval. Change in tobacco use status, from exclusive past 30 day use of any combustible-tobacco product to either exclusive past 30 day ENDS use, dual past 30 day use of ENDS and combustible-tobacco, or no past 30 day use of any tobacco, between the first two waves of an interval was used to predict onset of CVD between the second and third waves in the interval. CVD incidence was defined as a new self-report of being told by a health professional that they had congestive heart failure, stroke, or a myocardial infarction. Generalized estimating equation (GEE) analyses combined 10,548 observations across intervals from 7820 eligible respondents. Results: Overall, there were 191 observations of CVD among 10,548 total observations (1.7%, standard error (SE) = 0.2), with 40 among 3014 never users of tobacco (1.5%, SE = 0.3). In multivariable models, CVD incidence was not significantly different for any tobacco user groups compared to never users. There were 126 observations of CVD among 6263 continuing exclusive combustible-tobacco users (adjusted odds ratio [AOR] = 1.44; 95% confidence interval (CI) 0.87–2.39), 15 observations of CVD among 565 who transitioned to dual use (AOR = 1.85; 0.78–4.37), and 10 observations of CVD among 654 who quit using tobacco (AOR = 1.18; 0.33–4.26). There were no observations of CVD among 53 who transitioned to exclusive ENDS use. Conclusions: This study found no difference in CVD incidence by tobacco status over three 3 year intervals, even for tobacco quitters. It is possible that additional waves of PATH Study data, combined with information from other large longitudinal cohorts with careful tracking of ENDS use patterns may help to further clarify this relationship.