Kempf, W. | Search bioRxiv

bioRxiv2023

INKILN is a novel long noncoding RNA promoting vascular smooth muscle inflammation

BackgroundActivation of vascular smooth muscle cells (VSMCs) inflammation is vital to initiate vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is poorly understood. MethodsBulk RNA-seq in differentiated human VSMCs revealed a novel human-specific lncRNA called INflammatory MKL1 Interacting Long Noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation and human atherosclerosis and abdominal aortic aneurysm (AAA) samples. The transcriptional regulation of INKILN was determined through luciferase reporter system and chromatin immunoprecipitation assay. Both loss- and gain-of-function approaches and multiple RNA-protein and protein-protein interaction assays were utilized to uncover the role of INKILN in VSMC proinflammatory gene program and underlying mechanisms. Bacterial Artificial Chromosome (BAC) transgenic (Tg) mice were utilized to study INKLIN expression and function in ligation injury-induced neointimal formation. ResultsINKILN expression is downregulated in contractile VSMCs and induced by human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-{kappa}B site within its proximal promoter. INKILN activates the proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. Mechanistically, INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-{kappa}B pathway. INKILN depletion blocks ILI{beta}-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1, and the luciferase activity of an NF-{kappa}B reporter. Further, INKILN knockdown enhances MKL1 ubiquitination, likely through the reduced physical interaction with the deubiquitinating enzyme, USP10. INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in BAC Tg mice. ConclusionsThese findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human BAC Tg mice offer a novel and physiologically relevant approach for investigating human-specific lncRNAs under vascular disease conditions.

molecular biology↗

bioRxiv2021

MIF-2/D-DT is an atypical atherogenic chemokine that promotes advanced atherosclerosis and hepatic lipogenesis

Atherosclerosis is the underlying cause of cardiovascular diseases (CVDs) such as myocardial infarction and ischemic stroke. It is a lipid-triggered chronic inflammatory condition of the arterial vascular wall that is driven by various inflammatory pathways including atherogenic cytokines and chemokines. D-dopachrome tautomerase (D-DT), also known as macrophage migration inhibitory factor-2 (MIF-2), belongs to the MIF protein family, which is best known for its pathogenic role in a variety of inflammatory and immune conditions including CVDs. While MIF is well known as a promoter of atherogenic processes, MIF-2 has not been studied in atherosclerosis. Here, we investigated atherosclerosis in hyperlipidemic Mif-2-/-Apoe-/- mice and studied the role of MIF-2 in various atherogenic assays in vitro. We found that global Mif-2 deficiency as well as its pharmacological blockade by 4-CPPC protected against atherosclerotic lesion formation and vascular inflammation in models of early and advanced atherogenesis. On cellular level, MIF-2 promoted monocyte migration in 2D and 3D and monocyte arrest on aortic endothelial monolayers, promoted B-cell chemotaxis in vitro and B-cell homing in vivo, and increased macrophage foam cell formation. Dose curves and direct comparison in a 3D migration set-up suggest that MIF-2 may be a more potent chemokine than MIF for monocytes and B cells. We identify CXCR4 as a novel receptor for MIF-2. The evidence relies on a CXCR4 inhibitor, CXCR4 internalization experiments, MIF-2/CXCR4 binding studies by yeast-CXCR4 transformants, and fluorescence spectroscopic titrations with a soluble CXCR4 surrogate. Of note, Mif-2-/-Apoe-/- mice exhibited decreased plasma cholesterol and triglyceride levels, lower body weights, smaller livers, and profoundly reduced hepatic lipid accumulation compared to Apoe-/- mice. Mechanistic experiments in Huh-7 hepatocytes suggest that MIF-2 regulates the expression and activation of sterol-regulatory element binding protein-1 and -2 (SREBP-1, SREBP-2) to induce lipogenic downstream genes such as FASN and LDLR, while it attenuated the activation of the SREBP inhibiting AMPK pathway. Studies using receptor Inhibitors showed that SREBP activation and hepatic lipoprotein uptake by MIF-2 is mediated by both CXCR4 and CD74. Lastly and in line with a combined role of MIF-2 in vascular inflammation and hepatic lipid accumulation, MIF-2 was found to be profoundly upregulated in unstable human carotid plaques, underscoring a critical role for MIF-2 in advanced stages of atherosclerosis. Together, these data identify MIF-2 as a novel atherogenic chemokine and CXCR4 ligand that not only promotes lesion formation and vascular inflammation but also strongly affects hepatic lipogenesis in an SREBP-mediated manner, possibly linking atherosclerosis and hepatic steatosis.

immunology↗