Noncoding Mechanisms in NF-κB Driven Vascular Endothelial Cell Inflammation

Vascular inflammation is an integral step in the initiation and progression of atherosclerosis.Mediated primarily by the nuclear factor kappa B (NF-κB) signaling pathway, vascular endothelial cells (EC) produce pro-inflammatory cytokines and adhesion molecules which facilitate monocyte recruitment and their subsequent transmigration through the endothelium. Noncoding mechanisms at the chromatin and noncoding RNA levels have been implicated in the development of atherosclerosis. Understanding these noncoding processes is imperative as they can provide ways to control and fine-tune aberrant vascular inflammation. Therefore, this dissertation aimed to elucidate how EC inflammation is regulated by long noncoding RNAs (lncRNAs) and NF-κB-chromatin interactions. LncRNAs are a heterogeneous class of noncoding transcripts >200 base pairs in length, many of which have been reported to act in cis to regulate the expression of their neighboring genes. Work in this dissertation has identified interleukin-1β (IL-1β) regulated neighboring mRNA-lncRNA pairs which were defined based on localization on the same topologically associated domains (TADs), divergent transcription, and shared regulatory elements. This approach led to the discovery of lncRNA-CCL2 which is transcribed divergently to CCL2, encoding a chemokine that is involved in monocyte recruitment. Moreover, lncRNA-CCL2 was found to positively regulate CCL2 transcript levels via interaction with RNA binding proteins. The second half of the dissertation used evolutionary conservation and NF-κB chromatin interaction status as a way to delineate which NF-κB binding sites are likely to regulate gene expression. It was found that accessible chromatin regions bound by NF-κB prior to tumor necrosis factor alpha (TNFα) treatment showed the highest degree of evolutionary conservation and were enriched near TNFα up-regulated genes. Furthermore, deletion of several TNFα pre-bound and conserved NF-κB sites, particularly within the CCL2 super-enhancer, showed them to be important for gene regulation. Altogether, these findings increase our understanding of the noncoding mechanisms implicated in vascular inflammation and in the future could be targeted to selectively fine-tune the EC inflammatory response.Ph.D

Noncoding RNAs regulate NF-kappaB signaling to modulate blood vessel inflammation.

peer reviewedCardiovascular diseases such as atherosclerosis are one of the leading causes of morbidity and mortality worldwide. The clinical manifestations of atherosclerosis, which include heart attack and stroke, occur several decades after initiation of the disease and become more severe with age. Inflammation of blood vessels plays a prominent role in atherogenesis. Activation of the endothelium by inflammatory mediators leads to the recruitment of circulating inflammatory cells, which drives atherosclerotic plaque formation and progression. Inflammatory signaling within the endothelium is driven predominantly by the pro-inflammatory transcription factor, NF-kappaB. Interestingly, activation of NF-kappaB is enhanced during the normal aging process and this may contribute to the development of cardiovascular disease. Importantly, studies utilizing mouse models of vascular inflammation and atherosclerosis are uncovering a network of noncoding RNAs, particularly microRNAs, which impinge on the NF-kappaB signaling pathway. Here we summarize the literature regarding the control of vascular inflammation by microRNAs, and provide insight into how these microRNA-based pathways might be harnessed for therapeutic treatment of disease. We also discuss emerging areas of endothelial cell biology, including the involvement of long noncoding RNAs and circulating microRNAs in the control of vascular inflammation

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

peer reviewedThe transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis

Paradoxical Suppression of Atherosclerosis in the Absence of microRNA-146a

Rationale: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor kappa light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)-derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the miR-146a precursor have been associated with risk of coronary artery disease. Objective: To define the role of endogenous miR-146a during atherogenesis. Methods and Results: Paradoxically, Ldlr(-/-) (low-density lipoprotein receptor null) mice deficient in miR-146a develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in Ldlr(-/-);miR-146a(-/-) mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with Ldlr(-/-) mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of miR-146a in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking miR-146a in BM-derived cells. Furthermore, we identify sortilin-1 (Sort1), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a. Conclusions: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells

Summary: Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease. : Shikatani et al. demonstrate that the nuclear transcription factor c-Myb exacerbates experimental atherosclerosis directly through its effects on B lymphocytes. Paradoxically, c-Myb promotes B2 cell development yet limits numbers of IgM-producing antibody-secreting cells and levels of atheroprotective OxLDL-specific IgM antibodies