Laura S Shankman

KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis

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-CreERT2 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.

Detection of histone modifications at specific gene loci in single cells in histological sections

Chromatin immunoprecipitation assays have contributed greatly to our understanding of the role of histone modifications in gene regulation. However, they do not permit analysis with single-cell resolution, thus confounding analyses of heterogeneous cell populations. Here we present a method that permits visualization of histone modifications of single genomic loci with single-cell resolution in formaldehyde-fixed paraffin-embedded tissue sections based on combined use of in situ hybridization and proximity ligation assays. We show that dimethylation of lysine 4 of histone H3 (H3K4me2) at the MYH11 locus is restricted to the smooth muscle cell (SMC) lineage in human and mouse tissue sections and that the mark persists even in phenotypically modulated SMC in atherosclerotic lesions that show no detectable expression of SMC marker genes. This methodology has promise for broad applications in the study of epigenetic mechanisms in complex multicellular tissues in development and disease.

Cooperative Binding of KLF4, pELK-1, and HDAC2 to a G/C Repressor Element in the SM22α Promoter Mediates Transcriptional Silencing During SMC Phenotypic Switching In Vivo

Rationale: We previously identified conserved G/C Repressor elements in the promoters of most smooth muscle cell (SMC) marker genes and demonstrated that mutation of this element within the SM22&agr; promoter nearly abrogated repression of this transgene after vascular wire injury or within lesions of ApoE−/− mice. However, the mechanisms regulating the activity of the G/C Repressor are unknown, although we have previously shown that phenotypic switching of cultured SMC is dependent on Krupple-like factor (KLF)4. Objective: The goals of the present studies were to ascertain if (1) injury-induced repression of SM22&agr; gene after vascular injury is mediated through KLF4 binding to the G/C Repressor element and (2) the transcriptional repressor activity of KLF4 on SMC marker genes is dependent on cooperative binding with pELK-1 (downstream activator of the mitogen-activated protein kinase pathway) and subsequent recruitment of histone de-acetylase 2 (HDAC2), which mediates epigenetic gene silencing. Methods and Results: Chromatin immunoprecipitation (ChIP) assays were performed on chromatin derived from carotid arteries of mice having either a wild-type or G/C Repressor mutant SM22&agr; promoter-LacZ transgene. KLF4 and pELK-1 binding to the SM22&agr; promoter was markedly increased after vascular injury and was G/C Repressor dependent. Sequential ChIP assays and proximity ligation analyses in cultured SMC treated with platelet-derived growth factor BB or oxidized phospholipids showed formation of a KLF4, pELK-1, and HDAC2 multiprotein complex dependent on the SM22&agr; G/C Repressor element. Conclusions: Silencing of SMC marker genes during phenotypic switching is partially mediated by sequential binding of pELK-1 and KLF4 to G/C Repressor elements. The pELK-1-KLF4 complex in turn recruits HDAC2, leading to reduced histone acetylation and epigenetic silencing.

Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective.

Although somatic cell activation of the embryonic stem cell (ESC) pluripotency factor OCT4 has been reported, this previous work has been controversial and has not demonstrated a functional role for OCT4 in somatic cells. Here we demonstrate that smooth muscle cell (SMC)-specific conditional knockout of Oct4 in Apoe−/− mice resulted in increased lesion size and changes in lesion composition that are consistent with decreased plaque stability, including a thinner fibrous cap, increased necrotic core area, and increased intraplaque hemorrhage. Results of SMC-lineage-tracing studies showed that these effects were probably the result of marked reductions in SMC numbers within lesions and SMC investment within the fibrous cap, which may result from impaired SMC migration. The reactivation of Oct4 within SMCs was associated with hydroxymethylation of the Oct4 promoter and was hypoxia inducible factor-1α (HIF-1α, encoded by HIF1A) and Krüppel-like factor-4 (KLF4)-dependent. These results provide the first direct evidence that OCT4 has a functional role in somatic cells, and they highlight the potential role of OCT4 in normal and diseased somatic cells.

Erratum: Corrigendum: KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis

Evelyn Schmid, Stefan Neef, Christopher Berlin, Angela Tomasovic, Katrin Kahlert, Peter Nordbeck, Katharina Deiss, Sabrina Denzinger, Sebastian Herrmann, Erich Wettwer, Markus Weidendorfer, Daniel Becker, Florian Schäfer, Nicole Wagner, Süleyman Ergün, Joachim P Schmitt, Hugo A Katus, Frank Weidemann, Ursula Ravens, Christoph Maack, Lutz Hein, Georg Ertl, Oliver J Müller, Lars S Maier, Martin J Lohse & Kristina Lorenz Nat. Med. 21, 1298–1306 (2015); published online 19 October 2015; corrected after print 29 October 2015

Context-dependent compensation among phosphatidylserine-recognition receptors

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk−/−Bai1Tg) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk−/− mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk+/+ and Mertk−/− mice. Prior to the onset of photoreceptor degeneration, Mertk−/− mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.

Irradiation abolishes smooth muscle investment into vascular lesions in specific vascular beds

The long-term adverse effects of radiotherapy on cardiovascular disease are well documented. However, the underlying mechanisms responsible for this increased risk are poorly understood. Previous studies using rigorous smooth muscle cell (SMC) lineage tracing have shown abundant SMC investment into atherosclerotic lesions, where SMCs contribute to the formation of a protective fibrous cap. Studies herein tested whether radiation impairs protective adaptive SMC responses during vascular disease. To do this, we exposed SMC lineage tracing (Myh11-ERT2Cre YFP+) mice to lethal radiation (1,200 cGy) followed by bone marrow transplantation prior to atherosclerosis development or vessel injury. Surprisingly, following irradiation, we observed a complete loss of SMC investment in 100% of brachiocephalic artery (BCA), carotid artery, and aortic arch lesions. Importantly, this was associated with a decrease in multiple indices of atherosclerotic lesion stability within the BCA. Interestingly, we observed anatomic heterogeneity, as SMCs accumulated normally into lesions of the aortic root and abdominal aorta, suggesting that SMC sensitivity to lethal irradiation occurs in blood vessels of neural crest origin. Taken together, these results reveal an undefined and unintended variable in previous studies using lethal irradiation and may help explain why patients exposed to radiation have increased risk for cardiovascular disease.