Delphine Gomez

Smooth muscle cell phenotypic switching in atherosclerosis

Smooth muscle cells (SMCs) possess remarkable phenotypic plasticity that allows rapid adaptation to fluctuating environmental cues, including during development and progression of vascular diseases such as atherosclerosis. Although much is known regarding factors and mechanisms that control SMC phenotypic plasticity in cultured cells, our knowledge of the mechanisms controlling SMC phenotypic switching in vivo is far from complete. Indeed, the lack of definitive SMC lineage-tracing studies in the context of atherosclerosis, and difficulties in identifying phenotypically modulated SMCs within lesions that have down-regulated typical SMC marker genes, and/or activated expression of markers of alternative cell types including macrophages, raise major questions regarding the contributions of SMCs at all stages of atherogenesis. The goal of this review is to rigorously evaluate the current state of our knowledge regarding possible phenotypes exhibited by SMCs within atherosclerotic lesions and the factors and mechanisms that may control these phenotypic transitions.

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.

Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co‐localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF‐β in aneurysms related to Marfan (and Loeys‐Dietz) syndrome. Here we investigate TGF‐β signalling in normal and pathological human ascending aortic wall in syndromic and non‐syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF‐β1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF‐β1 mRNA level. The increase in stored TGF‐β1 was associated with enhanced LTBP‐1 protein and mRNA levels. These dysregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF‐β1 and LTBP‐1 staining and between their respective mRNA expressions. In parallel, decorin was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF‐β retention and Smad2 signalling in syndromic and non‐syndromic aneurysms of the ascending aorta. Copyright

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.

Epigenetic control of vascular Smooth Muscle Cells in Marfan and non-Marfan thoracic aortic aneurysms

Aims Human thoracic aortic aneurysms (TAAs) are characterized by extracellular matrix breakdown associated with progressive smooth muscle cell (SMC) rarefaction. These features are present in all types of TAA: monogenic forms [mainly Marfan syndrome (MFS)], forms associated with bicuspid aortic valve (BAV), and degenerative forms. Initially described in a mouse model of MFS, the transforming growth factor-β1 (TGF-β1)/Smad2 signalling pathway is now assumed to play a role in TAA of various aetiologies. However, the relation between the aetiological diversity and the common cell phenotype with respect to TGF-β signalling remains unexplained. Methods and results This study was performed on human aortic samples, including TAA [MFS, n = 14; BAV, n = 15; and degenerative, n = 19] and normal aortas (n = 10) from which tissue extracts and human SMCs and fibroblasts were obtained. We show that all types of TAA share a complex dysregulation of Smad2 signalling, independent of TGF-β1 in TAA-derived SMCs (pharmacological study, qPCR). The Smad2 dysregulation is characterized by an SMC-specific, heritable activation and overexpression of Smad2, compared with normal aortas. The cell specificity and heritability of this overexpression strongly suggest the implication of epigenetic control of Smad2 expression. By chromatin immunoprecipitation, we demonstrate that the increases in H3K9/14 acetylation and H3K4 methylation are involved in Smad2 overexpression in TAA, in a cell-specific and transcription start site-specific manner. Conclusion Our results demonstrate the heritability, the cell specificity, and the independence with regard to TGF-β1 and genetic backgrounds of the Smad2 dysregulation in human thoracic aneurysms and the involvement of epigenetic mechanisms regulating histone marks in this process.

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.

Impaired Splicing of Fibronectin Is Associated With Thoracic Aortic Aneurysm Formation in Patients With Bicuspid Aortic Valve

Objective—Thoracic aortic aneurysm is a common complication in patients with bicuspid aortic valve (BAV). Alternatively spliced extra domain A (EDA) of fibronectin (FN) has an essential role in tissue repair. Here we analyze the expression of FN spliceforms in dilated and nondilated ascending aorta of tricuspid aortic valve (TAV) and BAV patients. Methods and Results—The mRNA expression was analyzed in the ascending aorta by Affymetrix Exon arrays in patients with TAV (n=40) and BAV (n=69). EDA and extra domain B (EDB) expression was increased in dilated aorta from TAV patients compared with nondilated aorta (P<0.001 and P<0.05, respectively). In contrast, EDA expression was not increased in dilated aorta from BAV patients (P=0.25), whereas EDB expression was upregulated (P<0.01). The expression of EDA correlated with maximum aortic diameter in TAV (&rgr;=0.58) but not in BAV (&rgr;=0.15) patients. Protein analyses of EDA-FN showed concordant results. Transforming growth factor-&bgr; treatment influenced the splicing of FN and enhanced the formation of EDA-containing FN in cultured medial cells from TAV patients but not in cells derived from BAV patients. Gene set enrichment analysis together with multivariate and univariate data analyses of mRNA expression suggested that differences in the transforming growth factor-&bgr; signaling pathway may explain the impaired EDA inclusion in BAV patients. Conclusion—Decreased EDA expression may contribute to increased aneurysm susceptibility of BAV patients.

Early Atheroma-Derived Agonists of Peroxisome Proliferator–Activated Receptor-γ Trigger Intramedial Angiogenesis in a Smooth Muscle Cell–Dependent Manner

Rationale: Neovascularization favors intraplaque hemorrhage and plaque rupture. Development of therapeutic strategies against atheromatous angiogenesis requires elucidation of its initiating factors. Objective: We investigated the contribution of smooth muscle cells (SMCs) and atheroma-derived lipids to the initiation of atheroma-associated neoangiogenesis. Methods and Results: Forty human aortic segments, each harvested from a different donor, were classified as healthy or as bearing early atheromatous lesions, including fatty streaks and fibrolipidic atheroma, according to their histological features. Immunostaining for blood vessels and vascular endothelial growth factor-A (VEGF-A), as well as measurement of VEGF-A protein and mRNA levels by ELISA and real-time PCR, revealed that angiogenesis and VEGF-A production were enhanced in the medial layer of atheromatous aortas. The intramedial vessel density and invasiveness and the production of VEGF-A by medial SMCs were indeed increased in atheromatous aortas compared with healthy aortas. Furthermore, intimal layers of atheromatous aortas were enriched in soluble lipid mediators capable of inducing a sustained increase in VEGF-A production by medial SMCs, turning these cells into potent inducers of angiogenesis when incorporated into mouse Matrigel implants. Both effects were inhibited by the peroxisome proliferator–activated receptor-&ggr; inhibitor GW9662 and mimicked by its agonist, rosiglitazone. Conclusions: We show that VEGF-A production is upregulated in medial SMCs of human atheromatous aortas and that peroxisome proliferator–activated receptor-&ggr; agonists derived from early intimal lesions are likely to contribute to this phenotypic change. Our findings suggest that medial SMCs are central organizers of an angiogenic response initiated by the subendothelial accumulation of atherogenic lipids.

Modifications of Chromatin Dynamics Control Smad2 Pathway Activation in Aneurysmal Smooth Muscle Cells

Rationale: The activation of the Smad2 signaling pathway is thought to play an important role in human aneurysmal diseases as described by an important body of research. We previously showed that constitutive Smad2 activation is associated with Smad2 mRNA overexpression in aneurysmal vascular smooth muscle cells (VSMCs), which is dependent on epigenetic regulation of the SMAD2 promoter involving histone modifications. However, the underlying molecular mechanisms controlling Smad2 overexpression are currently unknown. Objective: The aim of the present study is to understand the mechanisms regulating the constitutive Smad2 overexpression in VSMCs by identification of the histone-modifying enzymes, transcription factors, and cofactors responsible for Smad2 promoter activation in aneurysmal disease. Methods and Results: This study was performed on medial tissue extracts and primary cultures of VSMCs of human thoracic aneurysms (n=17) and normal thoracic aortas (n=10). Here, we demonstrate that the activation of SMAD2 promoter is driven by the recruitment of a multipartner complex, including the transcription factor p53 and histone acetyltransferases. Remarkably, the transcriptional regulatory network of the SMAD2 promoter is dramatically altered in human aneurysmal VSMCs in vitro and in situ with a switch from Myc-dependent repression of SMAD2 in normal vessel to a p53-dependent activation of SMAD2 in aneurysms. Furthermore, histone acetyltransferases p300 and P300/CBP-associated protein play a major role in SMAD2 promoter activation by acting on histone acetylation, p53 recruitment, and acetylation. Conclusions: These results provide evidence for a major role of p53 and the complex composed of p300 and p300/CBP-associated protein in Smad2 activation in human aneurysmal VSMCs.

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta.

Borges L F, Gomez D, Quintana M, Touat Z, Jondeau G, Leclercq A, Meilhac O, Jandrot‐Perrus M, Gutierrez P S, Freymuller E, Vranckx R & Michel J‐B
(2010) Histopathology57, 917–932