Deficiency of lncRNA MERRICAL abrogates macrophage chemotaxis and diabetes-associated atherosclerosis.

Diabetes-associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in Ldlr-/- mice on a high-fat, high-sucrose-containing (HFSC) diet identifies a macrophage-enriched nuclear long noncoding RNA (lncRNA), MERRICAL (macrophage-enriched lncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. lncRNA-mRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines Ccl3 and Ccl4. MERRICAL-deficient macrophages exhibit lower Ccl3 and Ccl4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet-fed Ldlr-/- mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro-inflammatory responses. These findings unveil a regulatory mechanism whereby a macrophage-enriched lncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.

MicroRNA-mediated control of myocardial infarction in diabetes.

Diabetes mellitus is a global public health problem whose cases will continue to rise along with the progressive increase in obesity and the aging of the population. People with diabetes exhibit higher risk of cardiovascular complications, especially myocardial infarction (MI). microRNAs (miRNAs) are evolutionary conserved small non-coding RNAs involved in the regulation of biological processes by interfering in gene expression at the post-transcriptional level. Accumulating studies in the last two decades have uncovered the role of stage-specific miRNAs associated with key pathobiological events observed in the hearts of people with diabetes and MI, including cardiomyocyte death, angiogenesis, inflammatory response, myocardial remodeling, and myocardial lipotoxicity. A better understanding of the importance of these miRNAs and their targets may provide novel opportunities for RNA-based therapeutic interventions to address the increased risk of MI in diabetes.

Perivascular Fibrosis Is Mediated by a KLF10-IL-9 Signaling Axis in CD4+ T Cells.

BACKGROUND: Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Ang II (angiotensin II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. METHODS: TF (transcription factor) profiling was performed in peripheral blood mononuclear cells of hypertensive patients. CD4-targeted KLF10 (Kruppel like factor 10)-deficient (Klf10fl/flCD4Cre+; [TKO]) and CD4-Cre (Klf10+/+CD4Cre+; [Cre]) control mice were subjected to Ang II infusion. End point characterization included cardiac echocardiography, aortic imaging, multiorgan histology, flow cytometry, cytokine analysis, aorta and fibroblast transcriptomic analysis, and aortic single-cell RNA-sequencing. RESULTS: TF profiling identified increased KLF10 expression in hypertensive human subjects and in CD4+ T cells in Ang II-treated mice. TKO mice showed enhanced perivascular fibrosis, but not interstitial fibrosis, in aorta, heart, and kidney in response to Ang II, accompanied by alterations in global longitudinal strain, arterial stiffness, and kidney function compared with Cre control mice. However, blood pressure was unchanged between the 2 groups. Mechanistically, KLF10 bound to the IL (interleukin)-9 promoter and interacted with HDAC1 (histone deacetylase 1) inhibit IL-9 transcription. Increased IL-9 in TKO mice induced fibroblast intracellular calcium mobilization, fibroblast activation, and differentiation and increased production of collagen and extracellular matrix, thereby promoting the progression of perivascular fibrosis and impairing target organ function. Remarkably, injection of anti-IL9 antibodies reversed perivascular fibrosis in Ang II-infused TKO mice and C57BL/6 mice. Single-cell RNA-sequencing revealed fibroblast heterogeneity with activated signatures associated with robust ECM (extracellular matrix) and perivascular fibrosis in Ang II-treated TKO mice. CONCLUSIONS: CD4+ T cell deficiency of Klf10 exacerbated perivascular fibrosis and multi-organ dysfunction in response to Ang II via upregulation of IL-9. Klf10 or IL-9 in T cells might represent novel therapeutic targets for treatment of vascular or fibrotic diseases.

Isolation and culture of murine aortic cells and RNA isolation of aortic intima and media: Rapid and optimized approaches for atherosclerosis research.

BACKGROUND AND AIMS: Isolation of cellular constituents from the mouse aorta is commonly used for expression or functional analyses in atherosclerosis research. However, current procedures to isolate primary cells are difficult, inefficient, and require separate mice. RNA extraction from aortic intima and media for transcriptomic analysis is also considered difficult with mixed RNA yields. To address these gaps, we provide: 1) a rapid, efficient protocol to isolate and culture diverse cell types concomitantly from the mouse aorta using immunomagnetic cell isolation; and 2) an optimized aortic intimal peeling technique for efficient RNA isolation from the intima and media. METHODS AND RESULTS: Aortic cells were obtained using an enzymatic solution and different cell types were isolated by magnetic beads conjugated to antibodies targeting endothelial cells (CD31+), leukocytes (CD45+), and fibroblast cells (CD90.2+), and smooth muscle cells were isolated by negative selection. Our protocol allows the isolation of relatively large numbers of cells (10,000 cells per aorta) in a predictable manner with high purity (>90%) verified by cell-marker gene expression, immunofluorescence, and flow cytometry. These cells are all functionally active when grown in cell culture. We also provide a rapid method to collect aortic intima-enriched RNA from Ldlr-/- mice utilizing an intima peeling approach and assess transcriptomic profiling associated with accelerated lesion formation. CONCLUSIONS: This protocol provides an effective means for magnetic bead-based isolation of different cell types from the mouse aortic wall, and the isolated cells can be utilized for functional and mechanistic studies for a range of vascular diseases including atherosclerosis.

A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor.

Objective: Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. Approach and Results: We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr−/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-ß1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600­1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. Conclusions: The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.

Recurrent pulmonary embolism associated with deep venous thrombosis diagnosed as protein s deficiency owing to a novel mutation in PROS1: A case report.

RATIONALE: Protein S (PS) deficiency that can be inherited or acquired is an independent risk factor for venous thromboembolism (VTE). PATIENT CONCERNS: In this report, we present a case of recurrent pulmonary embolism (PE) and deep venous thrombosis (DVT) due to PS deficiency. DIAGNOSES: A 32-year-old male patient with significant decrease in PS activity was detected by laboratory tests. Genetic examination of the PROS1 gene showed a transition of G to T in exon 14 (c.1792 G>T, p.E598X), which was a paternal inherited heterozygous G1792T substitution in the laminin G-type repeat domain, generating a premature stop codon at Glu598. INTERVENTIONS: We considered that the inherited PS deficiency due to a PROS1 gene mutation may associate with recurrent VTE. The patient was suggested to have an extended anticoagulant therapy to avoid a severe VTE event. OUTCOMES: The patient was discharged home with continued oral anticoagulants and was still seen in clinic for follow-up. LESSONS: It is necessary for the young patient with recurrent idiopathic thrombosis to perform an inherited PS deficiency test and receive anticoagulant therapy for an extended period.

Treatment of distal splenic artery aneurysm by laparoscopic aneurysmectomy with end-to-end anastomosis: A case report.

RATIONALE: Splenic artery aneurysm (SAA) is the most common visceral artery aneurysm, while most SAAs are treated by endovascular or open procedures. PATIENT CONCERNS: Here we present a case of SAA treated by laparoscopic aneurysmectomy with end-to-end anastomosis. DIAGNOSES: A 40-year-old woman was incidentally found to have an asymptomatic distal SAA. CT scan revealed the SAA to be located at the hilum of the spleen, with a maximal diameter of 2.7 cm. INTERVENTIONS: To prevent sudden rupture, the patient received laparoscopic aneurysmectomy. During the operation, end-to-end anastomosis was also performed since a tortuous proximal splenic artery prevented delivery of the stent graft. OUTCOMES: The patient was ambulated 12 hours after surgery and discharged 5 days later. Postoperative recovery was smooth without hemorrhage, infarction, infection, or splenic artery thrombosis. At 10-month follow-up,no hemorrhage, aneurysm recurrence, spleen infarction, splenic artery stenosis, or thrombosis had occurred. LESSONS: Patients with distal SAA can be treated by laparoscopic aneurysmectomy with end-to-end anastomosis to preserve the spleen. The laparoscopic procedure is safe and feasible in the selected patients.

Inhibition of Receptor-Interacting Protein Kinase 1 with Necrostatin-1s ameliorates disease progression in elastase-induced mouse abdominal aortic aneurysm model.

Abdominal aortic aneurysm (AAA) is a common aortic disease with a progressive nature. There is no approved pharmacological treatment to effectively slow aneurysm growth or prevent rupture. Necroptosis is a form of programmed necrosis that is regulated by receptor-interacting protein kinases (RIPs). We have recently demonstrated that the lack of RIP3 in mice prevented aneurysm formation. The goal of the current study is to test whether perturbing necroptosis affects progression of existing aneurysm using the RIP1 inhibitors Necrostatin-1 (Nec-1) and an optimized form of Nec-1, 7-Cl-O-Nec-1 (Nec-1s). Seven days after aneurysm induction by elastase perfusion, mice were randomly administered DMSO, Nec-1 (3.2 mg/kg/day) and Nec-1s (1.6 mg/kg/day) via intraperitoneal injection. Upon sacrifice on day 14 postaneurysm induction, the aortic expansion in the Nec-1s group (64.12 ± 4.80%) was significantly smaller than that of the DMSO group (172.80 ± 13.68%) (P < 0.05). The mean aortic diameter of Nec-1 treated mice appeared to be smaller (121.60 ± 10.40%) than the DMSO group, though the difference was not statistically significant (P = 0.1). Histologically, the aortic structure of Nec-1s-treated mice appeared normal, with continuous and organized elastin laminae and abundant αActin-expressing SMCs. Moreover, Nect-1s treatment diminished macrophage infiltration and MMP9 accumulation and increased aortic levels of tropoelastin and lysyl oxidase. Together, our data suggest that pharmacological inhibition of necroptosis with Nec-1s stabilizes pre-existing aneurysms by diminishing inflammation and promoting connective tissue repair.

Receptor-interacting protein kinase 3 contributes to abdominal aortic aneurysms via smooth muscle cell necrosis and inflammation.

RATIONALE: Depletion of medial smooth muscle cell (SMC) is a major pathological characteristic of abdominal aortic aneurysm (AAA), although the mechanism by which these cells are eliminated remains incompletely understood. We reasoned that necroptosis, a recently described form of necrosis mediated by receptor-interacting protein kinase 3 (RIP3), may contribute to AAA pathology through the induction of SMC death and the significant production of inflammatory cytokines. OBJECTIVE: To test the hypothesis that RIP3-mediated necroptosis is actively involved in aneurysm pathogenesis. METHODS AND RESULTS: RIP3 and RIP1 levels were found to be elevated in human AAAs, most noticeably in SMCs. Elevations of RIP3 and SMC necrosis were also observed in the elastase-induced mouse model of AAAs. Deletion of one or both copies of Rip3 prevented AAA formation. By transplanting Rip3(+/-) aortae to Rip3(+/+) mice, we demonstrated that reduced Rip3 expression in arterial wall was the primary cause of aneurysm resistance. In vitro, adenoviral overexpression of RIP3 was sufficient to trigger SMC necroptosis. Protein kinase C-delta contributed to tumor necrosis factor-α-induced SMC necroptosis by regulating Rip3 expression. Furthermore, Rip3 deficiency impaired tumor necrosis factor-α-induced inflammatory gene expression in aortic SMCs, which was at least in part because of attenuation of p65 Ser536 phosphorylation. In vivo, the lack of RIP3 diminished activation of p65 in SMCs, implicating a necrosis independent function of RIP3 in aneurysms. CONCLUSIONS: Enhanced RIP3 signaling in aneurysmal tissues contributes to AAA progression by causing SMC necroptosis, as well as stimulating vascular inflammation, and therefore may serve as a novel therapeutic target for AAA treatment.

Murine abdominal aortic aneurysm model by orthotopic allograft transplantation of elastase-treated abdominal aorta.

OBJECTIVE: Murine models have proved instrumental in studying various aspects of abdominal aortic aneurysm (AAA), from identification of underlying pathophysiologic changes to the development of novel therapeutic strategies. In the current study, we describe a new model in which an elastase-treated donor aorta is transplanted to a recipient mouse and allowed to progress to aneurysm. We hypothesized that by transplanting an elastase-treated abdominal aorta of one genotype to a recipient mouse of a different genotype, one can differentiate pathophysiologic factors that are intrinsic to the aortic wall from those stemming from circulation and other organs. METHODS: Elastase-treated aorta was transplanted to the infrarenal abdominal aorta of recipient mice by end-to-side microsurgical anastomosis. Heat-inactivated elastase-treated aorta was used as a control. Syngeneic transplants were performed with use of 12-week-old C57BL/6 littermates. Transplant grafts were harvested from recipient mice on day 7 or day 14 after surgery. The aneurysm outcome was measured by aortic expansion, elastin degradation, proinflammatory cytokine expression, and inflammatory cell infiltration and compared with that produced with the established, conventional elastase infusion model. RESULTS: The surgical technique success rate was 75.6%, and the 14-day survival rate was 51.1%. By day 14 after surgery, all of the elastase-treated transplanted abdominal aortas had dilated and progressed to AAAs, defined as 100% or more increase in the maximal external diameter compared with that measured before elastase perfusion, whereas none of the transplanted aortas pretreated with inactive elastase became aneurysmal (percentage increase in maximum aortic diameter: 159.36% ± 23.27%, transplanted elastase, vs 41.46% ± 9.34%, transplanted inactive elastase). Aneurysm parameters, including elastin degradation and infiltration of macrophages and T lymphocytes, were found to be identical to those observed in the conventional elastase model. Quantitative polymerase chain reaction analysis revealed similarly increased levels of proinflammatory cytokines (relative changes of mRNA in the conventional elastase model vs transplant model: tumor necrosis factor α, 1.71 ± 0.27 vs 2.93 ± 0.86; monocyte chemoattractant protein 1, 2.36 ± 0.58 vs 2.87 ± 0.51; chemokine (C-C motif) ligand 5, 3.37 ± 0.92 vs 3.46 ± 0.83; and interferon γ, 3.09 ± 0.83 vs 5.30 ± 1.69). Using green fluorescent protein transgenic mice as donors or recipients, we demonstrated that a small quantity of mononuclear leukocytes in the transplant grafts bared the genotype of the donors. CONCLUSIONS: Transplanted elastase-treated abdominal aorta could develop to aneurysm in recipient mice. This AAA transplant model can be used to examine how the microenvironment of a transplanted aneurysmal aorta may be altered by the contributions of the "global" environment of the recipient.