Assessment of Elastase-Induced Murine Abdominal Aortic Aneurysms: Comparison of Ultrasound Imaging with In Situ Video Microscopy

Aims. The aim of this study was to definitively assess the validity of noninvasive high-frequency ultrasound (US) measurements of aortic luminal diameter (ALD) in a murine model of elastase-induced abdominal aortic aneurysm in comparison with in situ video microscopy (VM). Methods. C57BL/6 mice underwent transient perfusion of the aorta with either elastase (n = 20: Elastase group) or saline (n = 10: Sham). Unoperated mice (n = 10) were also studied. Results. ALD measurements by US had excellent linear correlation and absolute agreement with that by VM in both Control (unoperated or sham-operated mice) and elastase groups (r = 0.96, intraclass correlation coefficient (ICC) = 0.88 and r = 0.93, ICC = 0.92, resp.). Bland-Altman analysis of US compared with VM measurements in both groups indicated good agreement, however US measurements were slightly but significantly higher than VM measurements in the control group (mean bias 0.039 mm, P < .05). Linear regression analysis revealed excellent correlation between US and VM measurements in both groups. (R2 = 0.91 in Control group, R2 = 0.85 in elastase group.) The reliability of US measurements was also confirmed by ex vivo histological measurements. Conclusions. High-frequency US provides reliable ALD measurements in developing murine abdominal aortic aneurysms

Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression.

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease

Correlation of computed tomography with carotid plaque transcriptomes associates calcification with lesion-stabilization

Background and aims: Unstable carotid atherosclerosis causes stroke, but methods to identify patients and lesions at risk are lacking. We recently found enrichment of genes associated with calcification in carotid plaques from asymptomatic patients. Here, we hypothesized that calcification represents a stabilising feature of plaques and investigated how macro-calcification, as estimated by computed tomography (CT), correlates with gene expression profiles in lesions. Methods: Plaque calcification was measured in pre-operative CT angiographies. Plaques were sorted into high- and lowcalcified, profiled with microarrays, followed by bioinformatic analyses. Immunohistochemistry and qPCR were performed to evaluate the findings in plaques and arteries with medial calcification from chronic kidney disease patients. Results: Smooth muscle cell (SMC) markers were upregulated in high-calcified plaques and calcified plaques from symptomatic patients, whereas macrophage markers were downregulated. The most enriched processes in high-calcified plaques were related to SMCs and extracellular matrix (ECM) organization, while inflammation, lipid transport and chemokine signaling were repressed. These findings were confirmed in arteries with high medial calcification. Proteoglycan 4 (PRG4) was identified as the most upregulated gene in association with plaque calcification and found in the ECM, SMA+ and CD68+/TRAP + cells. Conclusions: Macro-calcification in carotid lesions correlated with a transcriptional profile typical for stable plaques, with altered SMC phenotype and ECM composition and repressed inflammation. PRG4, previously not described in atherosclerosis, was enriched in the calcified ECM and localized to activated macrophages and smooth muscle-like cells. This study strengthens the notion that assessment of calcification may aid evaluation of plaque phenotype and stroke risk.The European Union’s Horizon 2020/Marie Sklodowska-Curie grant agreement No 722609 (INTRICARE);Swedish Heart and Lung FoundationSwedish Research Council (K2009-65X-2233-01-3, K2013- 65X-06816-30-4, 349-2007-8703)Uppdrag Besegra Stroke (P581/ 2011-123)Stockholm County Council (ALF2011-0260, ALF-2011- 0279)Swedish Society for Medical ResearchTore Nilsson’s FoundationMagnus Bergvall’s FoundationKarolinska Institutet FoundationEuropean Commission (722609)Publishe

Guidelines for the functional annotation of microRNAs using the Gene Ontology

MicroRNA regulation of developmental and cellular processes is a relatively new field of study, and the available research data have not been organized to enable its inclusion in pathway and network analysis tools. The association of gene products with terms from the Gene Ontology is an effective method to analyze functional data, but until recently there has been no substantial effort dedicated to applying Gene Ontology terms to microRNAs. Consequently, when performing functional analysis of microRNA data sets, researchers have had to rely instead on the functional annotations associated with the genes encoding microRNA targets. In consultation with experts in the field of microRNA research, we have created comprehensive recommendations for the Gene Ontology curation of microRNAs. This curation manual will enable provision of a high-quality, reliable set of functional annotations for the advancement of microRNA research. Here we describe the key aspects of the work, including development of the Gene Ontology to represent this data, standards for describing the data, and guidelines to support curators making these annotations. The full microRNA curation guidelines are available on the GO Consortium wiki (http://wiki.geneontology.org/index.php/MicroRNA_GO_annotation_manual)

Functional investigation of the coronary artery disease gene SVEP1

A missense variant of the sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein 1 (SVEP1) is genome-wide significantly associated with coronary artery disease. The mechanisms how SVEP1 impacts atherosclerosis are not known. We found endothelial cells (EC) and vascular smooth muscle cells to represent the major cellular source of SVEP1 in plaques. Plaques were larger in atherosclerosis-prone Svep1 haploinsufficient (ApoE^{−/−}Svep1^{+/−}) compared to Svep1 wild-type mice (ApoE^{−/−}Svep1^{+/+}) and ApoE^{−/−}Svep1^{+/−} mice displayed elevated plaque neutrophil, Ly6C^{high} monocyte, and macrophage numbers. We assessed how leukocytes accumulated more inside plaques in ApoE^{−/−}Svep1^{+/−} mice and found enhanced leukocyte recruitment from blood into plaques. In vitro, we examined how SVEP1 deficiency promotes leukocyte recruitment and found elevated expression of the leukocyte attractant chemokine (C-X-C motif) ligand 1 (CXCL1) in EC after incubation with missense compared to wild-type SVEP1. Increasing wild-type SVEP1 levels silenced endothelial CXCL1 release. In line, plasma Cxcl1 levels were elevated in ApoE^{−/−}Svep1^{+/−} mice. Our studies reveal an atheroprotective role of SVEP1. Deficiency of wild-type Svep1 increased endothelial CXCL1 expression leading to enhanced recruitment of proinflammatory leukocytes from blood to plaque. Consequently, elevated vascular inflammation resulted in enhanced plaque progression in Svep1 deficiency

Aging-regulated TUG1 is dispensable for endothelial cell function

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function

Aging-regulated TUG1 is dispensable for endothelial cell function

The evolutionary conserved Taurine Upregulated Gene 1 (TUG1) is a ubiquitously expressed gene that is one of the highest expressed genes in human and rodent endothelial cells (ECs). We here show that TUG1 expression decreases significantly in aging mouse carotid artery ECs and human ECs in vitro, indicating a potential role in the aging endothelial vasculature system. We therefore investigated if, and how, TUG1 might function in aging ECs, but despite extensive phenotyping found no alterations in basal EC proliferation, apoptosis, barrier function, migration, mitochondrial function, or monocyte adhesion upon TUG1 silencing in vitro. TUG1 knockdown did slightly and significantly decrease cumulative sprout length upon vascular endothelial growth factor A stimulation in human umbilical vein endothelial cells (HUVECs), though TUG1-silenced HUVECs displayed no transcriptome-wide mRNA expression changes explaining this effect. Further, ectopic expression of the highly conserved and recently discovered 153 amino acid protein translated from certain TUG1 transcript isoforms did not alter angiogenic sprouting in vitro. Our data show that, despite a high expression and strong evolutionary conservation of both the TUG1 locus and the protein sequence it encodes, TUG1 does not seem to play a major role in basic endothelial cell function

Matricellular protein CCN3 mitigates abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease