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Abdominal aortic aneurysm (AAA) is a common cardiovascular disease with a strong correlation to smoking, although underlying mechanisms have been minimally explored. Electronic cigarettes (e-cigs) have gained recent broad popularity and can deliver nicotine at comparable levels to tobacco cigarettes, but effects on AAA development are unknown.We evaluated the impact of daily e-cig vaping with nicotine on AAA using two complementary murine models and found that exposure enhanced aneurysm development in both models and genders. E-cigs induced changes in key mediators of AAA development including cytokine chitinase-3-like protein 1 (CHI3L1/Chil1) and its targeting microRNA-24 (miR-24). We show that nicotine triggers inflammatory signaling and reactive oxygen species while modulating miR-24 and CHI3L1/Chil1 in vitro, and that Chil1 is crucial to e-cig-augmented aneurysm formation using a knockout model.In conclusion our work shows increased aneurysm formation along with augmented vascular inflammation in response to e-cig exposure with nicotine. Further we identify Chil1 as a key mediator in this context. Our data raise concerns regarding the potentially harmful long-term effects of e-cig nicotine vaping.Smoking is one of the most hazardous modifiable risk factors, with clear links to abdominal aortic aneurysm. E-cig vaping has displayed explosive growth in popularity. Intended for smoking cessation, it has been taken up by millions with no such clinical need, delivering nicotine addiction to new generations. The presumption that vaping is safer than tobacco overlooks the potential cardiovascular risks of nicotine. This study shows for the first time that inhaled e-cig nicotine vapor augments experimental AAA and aortic inflammation, suggests a mechanistic role for the cytokine Chil1/CHI3L1 and its regulator microRNA-24, and raises red flags regarding longitudinal e-cig safety.

Background: Tight junction (TJ) disruption and dysfunction are involved in the progression of arteriosclerosis. miR-501-3p regulates endothelial TJ protein-1, resulting in TJ disruption. Because exosomal microRNAs can travel to distant tissues and influence cell behavior, patients with elevated miR-501-3p may experience accelerated vascular disease progression secondary to miR-501-3p-induced reductions in TJ. This study investigated whether plasma exosome miR-501-3p levels are associated with vascular stiffness, an indicator for arteriosclerotic changes. Methods and Results: Fifty-one subjects (mean [±SD] age 70±8 years, 37% male) enrolled in a medical checkup program were recruited to the study. Brachial-ankle arterial pulse wave velocity (baPWV) and plasma exosome miR-501-3p expression were measured. Patients were divided into 2 groups depending on whether their miR-501-3p ∆Ct values were above (“High”; n=24) or below (“Low”; n=27) the cut-off levels determined by receiver operating characteristic (ROC) curve analysis. Median (interquartile range) baPWV levels were significantly higher in the miR-501-3p High than Low group (1,664 [1,496–1,859] vs. 1,450 [1,353–1,686] cm/s, respectively; P

The Mongolian gerbil has historically been useful for brain ischemia experiments, owing to the gerbil's uniquely underdeveloped circle of Willis (CoW). This led to a gerbil model of cochlear ischemia being generated in our unit. However, we have found that the usual severe hearing loss seen in this model was not being induced consistently in recent experiments using the MON/Jms/GbsSlc gerbil (the sole commercially available gerbil in Japan). We set out to evaluate the posterior communicating artery (PcomA) in MON/Jms/GbsSlc, to re-establish whether this strain is appropriate for ischemia models. Having found that this unique feature is often lost, we then attempted to breed for the characteristic absent PcomA. India-ink perfusion revealed that the percentage of intact bilateral PcomA ("communicating type") in the MON/Jms/GbsSlc gerbil was 57%; unilateral only ("unilateral communicating type") was 39%; and completely absent PcomA ("non-communicating type") was 4%. We were able to obtain few examples of the indigenous old aged Japanese UNG/Mz gerbil strain (at University of Miyazaki). Unfortunately, the pure UNG/Mz female was too elderly for mating. Therefore, selective breeding crosses between MON/Jms/GbsSlc and male UNG/Mz were carried out. After five generations of selective breeding, the percentage of non-communicating type gerbils was significantly higher in the newly generated strain, MON/Jms/SlcMz (F6 generation; 63%) than in the MON/Jms/GbsSlc gerbil. Bilateral common carotid artery occlusion surgery demonstrated that the cerebral blood flow was significantly reduced in MON/Jms/SlcMz compared with MON/Jms/GbsSlc (p 0.0001) and induced more hippocampal injuries in MON/Jms/SlcMz than in MON/Jms/GbsSlc (p 0.01). In conclusion, the commercially available MON/Jms/GbsSlc gerbil can easily regain PcomA, and we established a new gerbil strain (MON/Jms/SlcMz) displaying non-PcomA.

Patients with type 2 diabetes (T2D) are threatened by excessive cardiovascular morbidity and mortality. While accelerated arterial stiffening may represent a critical mechanistic factor driving cardiovascular risk in T2D, specific therapies to contain the underlying diabetic arterial remodeling have been elusive. The present translational study investigates the role of microRNA-29b (miR-29b) as a driver and therapeutic target of diabetic aortic remodeling and stiffening. Using a murine model (db/db mice), as well as human aortic tissue samples, we find that diabetic aortic remodeling and stiffening is associated with medial fibrosis, as well as fragmentation of aortic elastic layers. miR-29b is significantly downregulated in T2D and miR-29b repression is sufficient to induce both aortic medial fibrosis and elastin breakdown through upregulation of its direct target genes COL1A1 and MMP2 thereby increasing aortic stiffness. Moreover, antioxidant treatment restores aortic miR-29b levels and counteracts diabetic aortic remodeling. Concluding, we identify miR-29b as a comprehensive—and therefore powerful—regulator of aortic remodeling and stiffening in T2D that moreover qualifies as a (redox-sensitive) target for therapeutic intervention., Graphical Abstract, Stiffening of large arteries represents a significant complication in patients with diabetes mellitus resulting in high blood pressure levels and widespread organ damage (in particular in heart, brain, and kidneys). In this study we identify microRNA-29b as a comprehensive regulator and potential therapeutical target of diabetic arterial remodeling and stiffening.

Supplemental Digital Content is available in the text., Background: Abdominal aortic aneurysm (AAA) is an important cause of cardiovascular mortality; however, its genetic determinants remain incompletely defined. In total, 10 previously identified risk loci explain a small fraction of AAA heritability. Methods: We performed a genome-wide association study in the Million Veteran Program testing ≈18 million DNA sequence variants with AAA (7642 cases and 172 172 controls) in veterans of European ancestry with independent replication in up to 4972 cases and 99 858 controls. We then used mendelian randomization to examine the causal effects of blood pressure on AAA. We examined the association of AAA risk variants with aneurysms in the lower extremity, cerebral, and iliac arterial beds, and derived a genome-wide polygenic risk score (PRS) to identify a subset of the population at greater risk for disease. Results: Through a genome-wide association study, we identified 14 novel loci, bringing the total number of known significant AAA loci to 24. In our mendelian randomization analysis, we demonstrate that a genetic increase of 10 mm Hg in diastolic blood pressure (odds ratio, 1.43 [95% CI, 1.24–1.66]; P=1.6×10−6), as opposed to systolic blood pressure (odds ratio, 1.06 [95% CI, 0.97–1.15]; P=0.2), likely has a causal relationship with AAA development. We observed that 19 of 24 AAA risk variants associate with aneurysms in at least 1 other vascular territory. A 29-variant PRS was strongly associated with AAA (odds ratioPRS, 1.26 [95% CI, 1.18–1.36]; PPRS=2.7×10−11 per SD increase in PRS), independent of family history and smoking risk factors (odds ratioPRS+family history+smoking, 1.24 [95% CI, 1.14–1.35]; PPRS=1.27×10−6). Using this PRS, we identified a subset of the population with AAA prevalence greater than that observed in screening trials informing current guidelines. Conclusions: We identify novel AAA genetic associations with therapeutic implications and identify a subset of the population at significantly increased genetic risk of AAA independent of family history. Our data suggest that extending current screening guidelines to include testing to identify those with high polygenic AAA risk, once the cost of genotyping becomes comparable with that of screening ultrasound, would significantly increase the yield of current screening at reasonable cost.

Abdominal aortic aneurysm (AAA) is associated with the loss of vascular smooth muscle cells (SMCs) within the vessel wall. Direct delivery of therapeutic cells is challenging due to impaired mechanical integrity of the vessel wall. We hypothesized that porous collagen scaffolds can be an effective vehicle for the delivery of human-derived SMCs to the site of AAA. The purpose was to evaluate if the delivery of cell-seeded scaffolds can abrogate progressive expansion in a mouse model of AAA. Collagen scaffolds seeded with either primary human aortic SMCs or induced pluripotent stem cell derived-smooth muscle progenitor cells (iPSC-SMPs) had >80% in vitro cell viability and >75% cell penetrance through the scaffold's depth, while preserving smooth muscle phenotype. The cell-seeded scaffolds were successfully transplanted onto the murine aneurysm peri-adventitia on day 7 following AAA induction using pancreatic porcine elastase infusion. Ultrasound imaging revealed that SMC-seeded scaffolds significantly reduced the aortic diameter by 28 days, compared to scaffolds seeded with iPSC-SMPs or without cells (acellular scaffold), respectively. Bioluminescence imaging demonstrated that both cell-seeded scaffold groups had cellular localization to the aneurysm but a decline in survival with time. Histological analysis revealed that both cell-seeded scaffold groups had more SMC retention and less macrophage invasion into the medial layer of AAA lesions, when compared to the acellular scaffold treatment group. Our data suggest that scaffold-based SMC delivery is feasible and may constitute a platform for cell-based AAA therapy.

Significance: Both smoking and family history are major risk factors for abdominal aortic aneurysm (AAA). Smoking is a powerful modulator of DNA methylation, and nicotine alone can cause heritable epigenetic alterations in animal models and humans. Nicotine also increases aortic stiffness, predisposing towards AAA. We investigated the effects of parental nicotine exposure in mice on their offspring’s risk for experimental AAA and on transgenerational DNA methylation. Methods: Male and female Apo E-/- mice (F0) were exposed to subcutaneous nicotine (25 mg/kg/day) or saline infusion for 28 days. After treatment completion, mice were mated to untreated controls. F1 generation offspring underwent Angiotensin II infusion (1μg/kg/min) to induce AAA at age 10-weeks. Aneurysm growth was tracked via ultrasound over 28 days. Subgroups of F1 mice underwent ex-vivo pressure myography assessment of the abdominal aorta. Germ and aortic tissue from both F0 and F1 were evaluated using RRBS-Seq genome-wide DNA methylation analysis. Results: Parental nicotine augments model AAA formation, incidence, and rupture rates in their offspring. This effect was most significant in male offspring of nicotine-exposed females (vs. saline). Maternal nicotine exposure also increased F1 aortic stiffness. F0 nicotine exposure altered DNA methylation in multiple tissues, and led to numerous significant differentially methylated regions (DMRs) in their F1 offspring, many precisely conserved. Nicotine caused global DNA hypermethylation, altered maternally imprinted genes in F0 females, and had variable patterns depending on gender. Nicotine altered aortic methylation patterns in genes known to relate to AAA development, including lncRNAs/miRNAs. DMR pathway analysis revealed enrichment for transcription factors, suggesting that nicotine transgenerationally influences numerous genes through expression modulation. Conclusion: Nicotine exposure can augment experimental AAA growth and aortic stiffness across generations. These effects are accompanied by widespread epigenetic changes in germ tissue and aorta, including several key AAA modulator genes, with enrichment in transcription factors, including many targeting known AAA-related genes.

Inflammation plays a major role in the pathogenesis of pulmonary hypertension (PH). We sought to investigate the effects of a cell-based immunomodulation in a dysimmune model of PH. PH was induced in athymic nude rats using semaxinib (Su group

Background and aims: Hyperlipidemia is a suggested risk factor for abdominal aortic aneurysm (AAA). However, whether hyperlipidemia is causally involved in AAA progression remains elusive. Here, we tested the hypothesis that hyperlipidemia aggravates AAA formation in the widely used porcine pancreatic elastase (PPE) model of AAA in mice with varying levels of plasma lipids. Methods: Prior to PPE-surgery, 8-week-old male C57BL/6J mice (n = 32) received 1·1011 viral genomes of rAAV8-D377Y-mPcsk9 or control rAAV8 via the tail vein. Mice were fed either western type diet or regular chow. At baseline and during the 28 days following PPE-surgery, mice underwent weekly ultrasonic assessment of AAA progression. Experiments were repeated using Apolipoprotein E knockout (ApoE−/−) mice (n = 7) and wildtype C57BL/6J mice (n = 5). Results: At sacrifice, maximal intergroup plasma cholesterol and non-HDL/HDL ratio differences were >5-fold and >20-fold, respectively. AAA diameters expanded to 150% of baseline, but no intergroup differences were detected. This was verified in an independent experiment comparing 8-week-old male ApoE−/− mice with wildtype mice. Histological evaluation of experimental AAA lesions revealed accumulated lipid in neointimal and medial layers, and analysis of human AAA lesions (n = 5) obtained from open repair showed medial lipid deposition. Conclusions: In summary, we find that lipid deposition in the aortic wall is a feature of PPE-induced AAA in mice as well as human AAA lesions. Despite, our data do not support the hypothesis that hyperlipidemia contributes to AAA progression.

Objective Identification of undefined risk factors will be crucial for the development of therapeutic strategies in hearing impairment. Platelets are likely to affect the development of sudden sensorineural hearing loss, which is a primary risk factor for permanent hearing impairment. This implies that abnormal platelets might contribute to long-term hearing loss. This study investigated the role of platelets in the development of hearing impairment over a 5-year period. Methods This study was a retrospective cohort study and consisted of a population-based survey, which was performed for 1,897 participants in 2014 to 2019. To evaluate the effect of platelet level on hearing ability, the subjects were divided into two groups: a high-normal platelet group (25 ∼ 40 × 104 cells/μL) and a low-normal platelet group (15 ∼ 25 × 104 cells/μL). Subjects were defined as having hearing impairment when pure tone audiometry was over 25 dB HL in either ear (tested in 2017 and 2019). Incidence of hearing impairment was analyzed. Results Incidence of hearing impairment at low frequencies was significantly higher in the low-normal platelet group than in the high-normal group year over year. Low-normal platelet count associated with low-frequency hearing impairment (LFHI) incidence (odds ratio [OR], 2.34; 95% confidence interval [CI], 1.15-4.76). In the low-normal platelet group, subjects whose counts declined from baseline developed more LFHI than those whose counts increased over time. Further, decreasing platelets appeared to be an independent risk factor contributing to the incidence of LFHI (OR, 2.10; 95%CI, 1.09-4.06) in the low-normal platelet group. Conclusion Both a low-normal platelet and a declining platelet count were independently associated with the incidence of LFHI. Level of evidence 3 Laryngoscope, 131:E1287-E1295, 2021.

Objective— There are currently no effective treatments for the prevention of dementia associated with vascular cognitive impairment. MicroRNAs regulate gene expression at the post-transcriptional level and play key roles in vascular disorders. TNFα (tumor necrosis factor-α) regulates blood–brain barrier breakdown through modification of cerebral tight junctions. Here, we sought key TNFα-responsive microRNAs that might influence blood–brain barrier breakdown via cerebral tight junction disruption in vascular cognitive impairment. Approach and Results— Using a mouse model of vascular cognitive impairment, chronic cerebral hypoperfusion within the white matter was induced with bilateral common carotid artery stenosis (BCAS) surgery. TNFα gene expression was increased in white matter post-BCAS surgery, and TNFα stimulation decreased claudin-5, ZO-1 (tight-junction protein 1), and occludin gene expression in murine brain endothelial cells. In silico analysis predicted 8 candidate microRNAs as regulators of claudin-5, ZO-1, and occludin gene expression. Of these, only miR-501-3p was upregulated by TNFα in vitro and was upregulated in the white matter after BCAS surgery. Further, miR-501-3p directly bound to the 3′-untranslated region of human ZO-1 and downregulated transendothelial electric resistance. In vivo administration of a locked nucleic acid –modified antisense oligonucleotide versus miR-501-3p suppressed BCAS-induced reduction of ZO-1 gene expression and blood–brain barrier disruption within the white matter and significantly ameliorated working memory deficits after BCAS surgery. Conclusions— We here provide the first evidence that the TNFα–miR-501-3p–ZO-1 axis plays an important role in the pathogenesis of cerebral hypoperfusion–induced working memory deficits and white matter lesions, as a result of blood–brain barrier breakdown via tight junction disruption. Therapeutic manipulation of miR-501-3p holds promise for limiting vascular cognitive impairment progression.

Significance: Abdominal aortic aneurysm (AAA) is a potentially fatal condition, featuring the possibility of high-mortality rupture. To date, prophylactic surgery by means of open surgical repair or endovascular aortic repair at specific thresholds is considered standard therapy. Both surgical options hold different risk profiles of short- and long-term morbidity and mortality. Targeting early stages of AAA development to decelerate disease progression is desirable. Recent Advances: Understanding the pathomechanisms that initiate formation, maintain growth, and promote rupture of AAA is crucial to developing new medical therapeutic options. Inflammatory cells, in particular macrophages, have been investigated for their contribution to AAA disease for decades, whereas evidence on lymphocytes, mast cells, and neutrophils is sparse. Recently, there has been increasing interest in noncoding RNAs (ncRNAs) and their involvement in disease development, including AAA. Critical Issues: The current evidence on myeloid cells and ncRNAs in AAA largely originates from small animal models, making clinical extrapolation difficult. Although it is feasible to collect surgical human AAA samples, these tissues reflect end-stage disease, preventing examination of critical mechanisms behind early AAA formation. Future Directions: Gaining more insight into how myeloid cells and ncRNAs contribute to AAA disease, particularly in early stages, might suggest nonsurgical AAA treatment options. The utilization of large animal models might be helpful in this context to help bridge translational results to humans.

Objective Hypercholesterolemia, a risk factor in cognitive impairment, can be treated with statins. However, cognitive decline associated with "statins" (HMG-CoA reductase inhibitors) is a clinical concern. This pilot study investigated the effects of combining statins and regular exercise on cognitive function in coronary artery disease (CAD) patients with prior mild cognitive decline. Methods We recruited 43 consecutive CAD patients with mild cognitive decline. These patients were treated with a statin and weekly in-hospital aerobic exercise for 5 months. We measured serum lipids, exercise capacity, and cognitive function using the mini mental state examination (MMSE). Results Low-density lipoprotein cholesterol levels were significantly decreased, and maximum exercise capacity (workload) was significantly increased in patients with CAD and mild cognitive decline after treatment compared with before. Combined statin-exercise therapy significantly increased the median (range) MMSE score from 24 (22-25) to 25 (23-27) across the cohort (p65 years, sex, and presence of diabetes mellitus, a decrease in BMI during statin-exercise therapy was significantly correlated with an increase in the MMSE score (odds ratio: 4.57, 95% confidence interval: 1.05-20.0; p

Background The efficacy of selective Janus kinase 1/3 inhibitor R507 to prevent obliterative airway disease was analyzed in preclinical airway transplantation models. Methods Orthotopic trachea transplantations were performed between Lewis donors and Brown Norway rat recipients. Oral everolimus (4 mg/kg once per day) or oral respective inhaled R507 (60 mg/kg twice per day, each) was used for immunosuppression. Grafts were retrieved after 6 or 60 days. Toxicity and anti-inflammatory effects of R507 were analyzed on human airway epithelial cells. Results In 6-day animals, oral and inhaled R507 more potently diminished mononuclear graft infiltration than everolimus and preserved ciliated pseudostratified columnar respiratory epithelium. Everolimus and R507 similarly suppressed systemic cellular and humoral immune activation. In untreated rats, marked obliterative airway disease developed over 60 days. Oral and inhaled R507 was significantly more effective in reducing airway obliteration and preserved the morphology of the airway epithelium. Luciferase-positive donors revealed that a substantial amount of smooth muscle cells within the obliterative tissue was of donor origin. Only everolimus but not R507, adversely altered kidney function and lipid profiles. The R507 aerosol did not show airway toxicity in vitro but effectively suppressed activation of inflammatory signaling pathways induced by IL-1β. Conclusions The Janus kinase 1/3 inhibitor R507 is a very well-tolerated immunosuppressant that similarly diminished obliterative airway disease with systemic or inhaled administration.

We investigated the effects of variations in anesthesia exposure time prior to conducting anxiety response behavioral testing in sham controls from an experimental murine model. The staying time in the center area of the Open Field test in the “long exposure” group was significantly decreased compared to that of the “short exposure” group. Significant correlation was found between anesthesia time and the duration of staying time in the center area. We conclude that anesthesia time may have a significant impact on behavioral anxiety testing in this context, and advise careful control of this parameter in protocol optimization in related surgical animal models. Keywords: Anesthesia, Anxiety response, Rodent model

Background: Long noncoding RNAs have emerged as critical molecular regulators in various biological processes and diseases. Here we sought to identify and functionally characterize long noncoding RNAs as potential mediators in abdominal aortic aneurysm development. Methods: We profiled RNA transcript expression in 2 murine abdominal aortic aneurysm models, Angiotensin II (ANGII) infusion in apolipoprotein E–deficient ( ApoE −/− ) mice (n=8) and porcine pancreatic elastase instillation in C57BL/6 wild-type mice (n=12). The long noncoding RNA H19 was identified as 1 of the most highly upregulated transcripts in both mouse aneurysm models compared with sham-operated controls. This was confirmed by quantitative reverse transcription–polymerase chain reaction and in situ hybridization. Results: Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) in vivo, significantly limited aneurysm growth in both models. Upregulated H19 correlated with smooth muscle cell (SMC) content and SMC apoptosis in progressing aneurysms. Importantly, a similar pattern could be observed in human abdominal aortic aneurysm tissue samples, and in a novel preclinical LDLR −/− (low-density lipoprotein receptor) Yucatan mini-pig aneurysm model. In vitro knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, whereas overexpression of H19 had the opposite effect. Notably, H19-dependent apoptosis mechanisms in SMCs appeared to be independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array identified hypoxia-inducible factor 1α as the main downstream effector. Increased SMC apoptosis was associated with cytoplasmic interaction between H19 and hypoxia-inducible factor 1α and sequential p53 stabilization. Additionally, H19 induced transcription of hypoxia-inducible factor 1α via recruiting the transcription factor specificity protein 1 to the promoter region. Conclusions: The long noncoding RNA H19 is a novel regulator of SMC survival in abdominal aortic aneurysm development and progression. Inhibition of H19 expression might serve as a novel molecular therapeutic target for aortic aneurysm disease.

Objective— Recruitment of immunologic competent cells to the vessel wall is a crucial step in formation of abdominal aortic aneurysms (AAA). Innate immunity effectors (eg, macrophages), as well as mediators of adaptive immunity (eg, T cells), orchestrate a local vascular inflammatory response. IL-10 (interleukin-10) is an immune-regulatory cytokine with a crucial role in suppression of inflammatory processes. We hypothesized that an increase in systemic IL-10-levels would mitigate AAA progression. Approach and Results— Using a single intravenous injection protocol, we transfected an IL-10 transcribing nonimmunogenic minicircle vector into the Ang II (angiotensin II)-ApoE −/− infusion mouse model of AAA. IL-10 minicircle transfection significantly reduced average aortic diameter measured via ultrasound at day 28 from 166.1±10.8% (control) to 131.0±5.8% (IL-10 transfected). Rates of dissecting AAA were reduced by IL-10 treatment, with an increase in freedom from dissecting AAA from 21.5% to 62.3%. Using flow cytometry of aortic tissue from minicircle IL-10-treated animals, we found a significantly higher percentage of CD4 + /CD25 + /Foxp3 (forkhead box P3) + regulatory T cells, with fewer CD8 + /GZMB + (granzyme B) cytotoxic T cells. Furthermore, isolated aortic macrophages produced less TNF-α (tumor necrosis factor-α), more IL-10, and were more likely to be MRC1 (mannose receptor, C type 1)–positive alternatively activated macrophages. These results concurred with gene expression analysis of lipopolysaccharide-stimulated and Ang II-primed human peripheral blood mononuclear cells. Conclusions— Taken together, we provide an effective gene therapy approach to AAA in mice by enhancing antiinflammatory and dampening proinflammatory pathways through minicircle-induced augmentation of systemic IL-10 expression.

Background: Long noncoding RNAs (lncRNAs) have emerged as critical molecular regulators in various biological processes and diseases. Here we sought to identify and functionally characterize lncRNAs as potential mediators in abdominal aortic aneurysm (AAA) development. Methods and results: We profiled RNA transcript expression in two murine AAA models, Angiotensin II (ANGII) infusion in ApoE-/- mice ( n =10) and porcine pancreatic elastase (PPE) instillation in C57BL/6 wildtype mice ( n =12). The lncRNA H19 was identified as one of the most highly up-regulated transcripts in both mouse aneurysm models compared to sham-operated controls. This was confirmed by qRT-PCR and in situ hybridization. Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) in vivo , significantly limited aneurysm growth in both models. Upregulated H19 correlated with smooth muscle cell (SMC) content and SMC apoptosis in progressing aneurysms. Importantly, a similar pattern could be observed in human AAA tissue samples, and in a novel preclinical LDLR-/- Yucatan mini-pig aneurysm model. In vitro knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, while overexpression of H19 had the opposite effect. Notably, H19-dependent apoptosis mechanisms in SMCs appeared to be independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array identified hypoxia-inducible factor 1-alpha (HIF1α) as the main downstream effector. Increased SMC apoptosis was associated with cytoplasmic interaction between H19 and HIF1α and sequential p53 stabilization. Additionally, H19 induced transcription of HIF1α via recruiting specificity protein 1 (Sp1) transcription factor to the promoter region. Conclusions: The lncRNA H19 is a novel regulator of SMC survival in AAA development and progression. Inhibition of H19 expression might serve as a novel molecular therapeutic target for aortic aneurysm disease.

Rationale: Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown. Objective: To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism. Methods and Results: Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b . These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b -deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor β ( TGF β) signaling in cultured cyclin-dependent kinase inhibitor 2B ( CDKN2B )–deficient cells, as well as TGF β 1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7 , which promotes downstream TGF β activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGF β 1-induced-1 , which is a TGF β-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. Conclusions: These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGF β signaling and hypoxic neovessel maturation.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among patients with diabetes mellitus (DM). DM can lead to multiple cardiovascular complications, including coronary artery disease (CAD), cardiac hypertrophy, and heart failure (HF). HF represents one of the most common causes of death in patients with DM and results from DM-induced CAD and diabetic cardiomyopathy. Oxidative stress is closely associated with the pathogenesis of DM and results from overproduction of reactive oxygen species (ROS). ROS overproduction is associated with hyperglycemia and metabolic disorders, such as impaired antioxidant function in conjunction with impaired antioxidant activity. Long-term exposure to oxidative stress in DM induces chronic inflammation and fibrosis in a range of tissues, leading to formation and progression of disease states in these tissues. Indeed, markers for oxidative stress are overexpressed in patients with DM, suggesting that increased ROS may be primarily responsible for the development of diabetic complications. Therefore, an understanding of the pathophysiological mechanisms mediated by oxidative stress is crucial to the prevention and treatment of diabetes-induced CVD. The current review focuses on the relationship between diabetes-induced CVD and oxidative stress, while highlighting the latest insights into this relationship from findings on diabetic heart and vascular disease.

Aortic aneurysms represent a major public health burden, and currently have no medical treatment options. The pathophysiology behind these aneurysms is complex and variable, depending on location and underlying cause, and generally involves progressive dysfunction of all elements of the aortic wall. Changes in smooth muscle behavior, endothelial signaling, extracellular matrix remodeling, and to a variable extent inflammatory signaling and cells, all contribute to the dilation of the aorta, ultimately resulting in high mortality and morbidity events including dissection and rupture. A large number of researchers have identified non-coding RNAs as crucial regulators of aortic aneurysm development, both in humans and in animal models. While most work to-date has focused on microRNAs, intriguing information has also begun to emerge regarding the role of long-non-coding RNAs. This review summarizes the currently available data regarding the involvement of non-coding RNAs in aneurysmal aortopathies. Going forward, these represent key potential therapeutic targets that might be leveraged in the future to slow or prevent aortic aneurysm formation, progression and rupture.

Objective We assessed outcomes of open surgical venous thrombectomy with temporary arteriovenous fistula, and the procedure’s effect on health-related quality of life. Method We retrospectively analyzed 48 (26 at long-term) patient medical records. Mortality rates, patency, and risk of post-thrombotic syndrome were analyzed using Kaplan–Meier estimation. The association between risk factors/coagulation disorders and patency/post-thrombotic syndrome along with patient health-related quality of life at long-term was analyzed employing various statistical methods. Results Patient one-year survival rate was 93 ± 4% and primary one-year patency rate was 89 ± 5% (secondary one-year patency rate 97 ± 3%). Freedom from post-thrombotic syndrome after eight years was 80 ± 12% (post-thrombotic syndrome rate 20 ± 12%). Health-related quality of life was impaired vs. normative data in the physical and social subscales, and in the mental component score ( p Conclusions Open surgical venous thrombectomy appears safe compared with literature-reported outcomes in similar patients using alternative approaches. Iliofemoral deep vein thrombosis impairs physical, social, and mental health-related quality of life.

Background: Dementia cases are increasing as the population ages, leading to increased financial costs. Several neuronal diseases including ischemic and hemorrhagic stroke involve cerebrovascular injury or pathophysiology. Cerebrovascular injury is closely tied to blood brain barrier (BBB) disruption. Many studies have shown a significant association between BBB dysfunction and neurological diseases. Therefore, an understanding of the molecular mechanisms which regulate BBB permeability and disruption is essential for establishing future therapeutic strategies to alter dementia disease progression related to cerebrovascular injury,so-called vascular cognitive impairment (VCI). microRNAs (miRs) are small noncodingRNAs that regulate gene expression through targeting of mRNA transcripts.miRs have been implicated in the development and progression of various illnesses, including vascular disease. However, the role of miRs in BBB breakdown or permeability and VCI development has not yet been well clarified. Methods: Research content related to the origins of VCI and the role of the BBB in pathologic development and therapeutic targeting are reviewed, including current relevant animal models. We draw from the published literature regarding microRNA candidates that are associated with modulation of BBB structure and function. Results: In this review, we summarize the current knowledge about VCI, explore the potential role of miRs in BBB breakdown and VCI progression, and identify potential candidate miRs for development of new treatment strategies. Conclusion: miRs constitute a promising novel avenue as future therapeutic options for alteration of both BBB permeability and development of VCI.

Background: Abdominal aortic aneurysm (AAA) is defined as a permanent dilation of the abdominal aorta that is highly lethal in case of rupture. Current therapeutic approaches are limited to open surgery or stent-based endovascular aortic repair (EVAR) to exclude large sized AAA (>5.5 cm) from the circulation. However, to date there is no effective therapy to prevent small AAA formation and progression. Nonetheless, options for small AAA disease are highly desirable as even small AAA may rupture and app. 70% of all small AAA will grow and eventually require surgical repair. Objective: Building on our previous experimental research findings we present a novel therapeutic approach for early AAA intervention. Methods and Results: Our recent biomechanical studies in a murine model of AAA development (elastase model) as well as in human aortae indicate that early AAA growth is critically driven by a stiffness gradient between a stiff AAA segment and the adjacent more compliant aorta. As a promising therapeutic intervention, we found that stiffening of the AAA-adjacent aorta (by external glue application) was sufficient to stop murine AAA formation. In a translational approach we now aim to develop an intravascular stent prototype that is deployed in the neck region of a developing AAA to increase the mechanical stiffness of the AAA-adjacent aorta. We hypothesize that this intervention will decrease the aortic stiffness gradient towards the AAA segment – thereby preventing further AAA growth. Stent prototypes with varying designs will be tested in a pig model of AAA disease that closely resembles the human anatomic situation. Conclusion: This project demonstrates a highly promising opportunity to directly translate novel pathomechanistic insights into AAA pathobiology from bench to bedside application and stop early AAA progression in humans.

Background: Arterial stiffness is a major risk factor for various cardiovascular diseases and contributes to the development of abdominal aortic aneurysms (AAA). In this context, differential aortic stiffening of adjacent aortic segments increases aortic wall stress and accelerates the disease. Smoking is a major risk factor for AAA, in part due to nicotine. In this study, we investigated aortic stiffening of the thoracic and abdominal aorta and analyzed stiffness-related gene expression. Methods: 36 WT mice (C57BL/6) mice were infused with nicotine or PBS using osmotic mini pumps for 42 days. Thoracic segment (TS) and abdominal segment (AS) aortic stiffness were analyzed using ultrasound (M-Mode and PW). TS and AS were further investigated by ex vivo myograph measurements. Gene expression for TMIP2, MT1-MMP, MMP2, collagen type I and type III was performed for both segments separately. Results: Myograph measurements revealed increased strain within the AS after 2 weeks (p Discussion: Aortic stiffening in response to nicotine varies between the TS and AS segment over a time course of 6 weeks. This leads to an increased stiffness gradient between the TS and AS. Gene expression changes in stiffness-related genes occurred in response to nicotine, although no difference appeared between the segments. We conclude that the difference in stiffness development for TS and AS could be based on a different basic morphological structure involving elastin and collagen load, and that these responses may in part explain nicotine’s role in promoting AAA.

Background: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators in various biological processes and diseases. Here we sought to identify and functionally characterize the lncRNA H19 as a novel regulator in abdominal aortic aneurysm development. Methods and Results: We profiled RNA transcript expression in two murine abdominal aortic aneurysm models, Angiotensin II (ANGII) infusion in ApoE-/- mice and porcine pancreatic elastase (PPE) instillation in C57BL/6 wildtype mice. The lncRNA H19 was identified as one of the most highly up-regulated transcripts in both mouse aneurysm models compared to sham-operated controls. This was confirmed by qRT-PCR and in situ hybridization. Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) in vivo , significantly limited aneurysm growth in both models. Upregulated H19 correlated well with smooth muscle cell (SMC) content and its apoptosis in progressing aneurysms. Of importance, a similar pattern could be observed in human AAA tissue samples, and in a novel preclinical LDLR-/- Yucatan mini-pig aneurysm model. In vitro knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, while overexpression of H19 had the opposite effect. Interestingly, H19-dependent cell fate decisions in SMCs appeared independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array and proteomic analysis revealed the hypoxia-inducible factor 1-alpha (HIF1A) and interleukin 1 receptor-like 2 (IL1RL2) as the main downstream effectors being regulated via H19. Conclusion: The lncRNA H19 is a novel regulator of SMC survival and aortic inflammation in AAA development and progression. Ultimately inhibition of H19 expression might serve as a novel molecular therapeutic target for aneurysm disease. Key words: Long noncoding RNAs (lncRNAs); abdominal aortic aneurysm; smooth muscle cells (SMCs); Inflammation

Dementia is one of the greatest public health concerns for the modern aging world. Over the last decade, most researchers developing new therapeutic strategies for dementia have focused on amyloid-β. In contrast, numerous recent studies have indicated that vascular risk factors are associated with various forms of dementia, and that in fact most forms of dementia can be considered an extension of vascular disease. Accordingly, it is sensible to pursue treatment approaches that focus on the blood vessels. Blood-brain barrier (BBB) disruptions in the white matter of patients with vascular cognitive impairment (VCI) have been observed using imaging analysis, and might be potential targets for novel VCI treatment. Tight junctions between cerebral endothelial cells play an important role in the function of the BBB, and recent studies have demonstrated the essential role of microRNAs in regulating tight junctions. Further elucidation of the mechanisms of tight junction-disruption in dementia are likely to lead to promising novel treatments. In this article, we summarize current knowledge regarding microRNAs and vascular cognitive impairment and the possibility of utilizing microRNAs as biomarkers for BBB dysfunction, and seek to envision future therapeutic strategies.

Objective Inferior vena cava thrombosis is rare, but patients are at high risk for development of a post-thrombotic syndrome (PTS) in the long term. Surgical approaches include indirect transfemoral venous thrombectomy (iTFVT) and direct open venous thrombectomy (dOVT). This study reports patient outcomes after iTFVT and dOVT for inferior vena cava thrombosis covering a 25-year follow-up period. Methods The study period was from January 1, 1982, to December 31, 2013. Data were retrieved from archived medical records, and patients were invited for a detailed phlebologic follow-up examination (DPFE). Health-related quality of life was assessed with the 36-Item Short Form Health Survey questionnaire. Patient survival, patency rates, and freedom from PTS were calculated using Kaplan-Meier estimation with log-rank testing. The χ2 test with Yates continuity correction and logistic regression analysis were applied to identify associations between risk factors or coagulation disorders, mortality, and PTS. Results Complete medical records were available for 152 patients. Patients' 5-year survival was 91% ± 3%, and 5-year primary and secondary patency rates were 80% ± 3% and 94% ± 2%. Freedom from PTS after 25 years was 84% ± 6%. No differences for patient survival, patency rates, or freedom from PTS were identified between iTFVT, dOVT, and a combination of both procedures. Antithrombin III deficiency was the most common coagulation disorder, and patients' physical function and social function were impaired compared with those found in German normative data (P Conclusions Open surgical venous thrombectomy is safe and delivers satisfying short- and long-term outcomes compared with endovascular approaches. It remains valuable for patients who are not eligible for other interventional therapies.

Significance: Arterial blood vessels functionally and structurally adapt to altering hemodynamic forces in order to accommodate changing needs and to provide stress homeostasis. This ability is achieved at the cellular level by converting mechanical stimulation into biochemical signals (i.e., mechanotransduction). Physiological mechanical stress helps maintain vascular structure and function, whereas pathologic or aberrant stress may impair cellular mechano-signaling, and initiate or augment cellular processes that drive disease. Recent Advances: Reactive oxygen species (ROS) may represent an intriguing class of mechanically regulated second messengers. Chronically enhanced ROS generation may be induced by adverse mechanical stresses, and is associated with a multitude of vascular diseases. Although a causal relationship has clearly been demonstrated in large numbers of animal studies, an effective ROS-modulating therapy still remains to be established by clinical studies. Critical Issues and Future Directions: This review article focuses on the role of various mechanical forces (in the form of laminar shear stress, oscillatory shear stress, or cyclic stretch) as modulators of ROS-driven signaling, and their subsequent effects on vascular biology and homeostasis, as well as on specific diseases such as arteriosclerosis, hypertension, and abdominal aortic aneurysms. Specifically, it highlights the significance of the various NADPH oxidase (NOX) isoforms as critical ROS generators in the vasculature. Directed targeting of defined components in the complex network of ROS (mechano-)signaling may represent a key for successful translation of experimental findings into clinical practice. Antioxid. Redox Signal. 20, 914–928.

The contribution of abdominal aortic aneurysm (AAA) disease to human morbidity and mortality has increased in the aging, industrialized world. In response, extraordinary efforts have been launched to determine the molecular and pathophysiological characteristics of the diseased aorta. This work aims to develop novel diagnostic and therapeutic strategies to limit AAA expansion and, ultimately, rupture. Contributions from multiple research groups have uncovered a complex transcriptional and post-transcriptional regulatory milieu, which is believed to be essential for maintaining aortic vascular homeostasis. Recently, novel small noncoding RNAs, called microRNAs, have been identified as important transcriptional and post-transcriptional inhibitors of gene expression. MicroRNAs are thought to “fine tune” the translational output of their target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. With the discovery that microRNAs act as powerful regulators in the context of a wide variety of diseases, it is only logical that microRNAs be thoroughly explored as potential therapeutic entities. This current review summarizes interesting findings regarding the intriguing roles and benefits of microRNA expression modulation during AAA initiation and propagation. These studies utilize disease-relevant murine models, as well as human tissue from patients undergoing surgical aortic aneurysm repair. Furthermore, we critically examine future therapeutic strategies with regard to their clinical and translational feasibility.

Background: Chronic mesenteric ischemia (CMI) is a rare disease. Open treatment (OT) remains a valuable treatment option. We analyzed patient outcomes after OT and investigated health-related quality of life (HRQoL). Methods: Data were analyzed retrospectively. The investigation period was from January 1, 2001, to December 31, 2014. We investigated mortality and patency rates using Kaplan-Meier analysis. HRQoL was measured using a 36-item health survey. Various statistical methods were employed. Results: A total of 100 patients (celiac trunk [TC: n = 23], superior mesenteric artery [SMA: n = 26], or both [n = 51]) were included. Median follow-up was 5 ± 35 months. One-year survival rate for TC was 75 ± 11%, for SMA: 79 ± 10%, and for both: 96 ± 3%. TC 5-year survival was 75 ± 11% (SMA: 57 ± 16%: both: 80 ± 8%). Obesity and the length of hospital stay were independently associated with patient survival (p < 0.05). Primary 1-year patency rate was 60 ± 13% for TC (SMA: 86 ± 10%; both: 71 ± 8%) and secondary 1-year patency rate was 84 ± 9% for TC (SMA: 100%; both: 79 ± 7%). HRQoL was inferior compared to the German normative data (p < 0.05). Conclusion: CMI overlaps between gastrointestinal and vascular surgery. OT is safe, and simultaneous revascularization of the TC and the SMA does not affect mortality. Patients would not necessarily benefit from OT in terms of HRQoL.

In this study we investigated how hemostats such as oxidized regenerated cellulose (ORC, TABOTAMP) and oxidized non-regenerated cellulose (ONRC, RESORBA CELL) influence local cellular behavior and contraction of the extracellular matrix (ECM). Human stromal fibroblasts were inoculated in vitro with ORC and ONRC. Cell proliferation was assayed over time and migration was evaluated by Live Cell imaging microscopy. Fibroblasts grown in collagen-gels were treated with ORC or ONRC and ECM contraction was measured utilizing a contraction assay. An absolute pH decline was observed with both ORC and ONRC after 1 hour. Mean daily cell proliferation, migration and matrix contraction were more strongly inhibited by ONRC when compared with ORC (p in vivo experiments to clarify the clinical implications for hemostat use in post-surgical wound healing.

Background: Arterial stiffening is a hallmark of vascular aging and may constitute a critical mechanism linking age to increased cardiovascular risk. However, up to now there is no therapy available to efficiently and specifically target age-related arterial stiffening. We recently identified the osteogenic transcription factor Runx2 as an inducer of diabetic arterial stiffness. Objective: The present study investigated the role of Runx2 in the setting of age-related arterial stiffness. Methods and Results: Aortic stiffness – quantified by ex vivo mechanical testing (pressure myography) – was markedly increased in 1-year old male C57Bl/6 mice compared to young (10 week-old) controls. At the same time, Runx2 was aberrantly upregulated in the medial layer of aged aortae, coming along with significant medial fibrosis. Additionally, we detected increased aortic expression of interleukin 6 ( Il6 ) – a key cytokine involved in vascular “inflammaging”. Mechanistically, we found IL-6-induced RUNX2 expression in aortic smooth muscle cells (SMCs) via a NFkB-dependent pathway. Conclusion: In conclusion this study suggests Runx2 as a potential mediator of age-related arterial fibrosis and stiffness warranting further interventional/therapeutic studies.

Background: Accelerated arterial stiffening is a complication of diabetes mellitus and associated with the development of hypertension. Arterial stiffening results from extensive extracellular matrix remodeling (elastin breakdown, collagen accumulation). MicroRNA miR-29b directly regulates the expression of genes governing fibrosis (such as COL1A1, COL3A1) and elastin breakdown ( MMP2, MMP9 ). However, its impact on aortic stiffness is unclear. Objective: This study was designed to investigate the role of miR-29b as potential mediator of diabetic aortic stiffening. Methods and Results: Serial ex vivo mechanical testing of the thoracic aorta and volume-pressure recording (VPR) based tail-cuff blood pressure measurements revealed that aortic stiffening precedes blood (pulse) pressure elevations in diabetic db/db mice. Vascular stiffening was accompanied by increased elastin fragmentation and collagen deposition (EvG and Picrosirius Red staining). qRT-PCR, in-situ hybridization and immunohistochemistry revealed decreased expression of miR-29b and de-repression of target genes ( Col1A1, COL3A1, MMP2, MMP9 ) in db/db mice compared to controls. Investigating the mechanistic significance of miR-29b for arterial stiffening, forced downregulation of miR-29b (via systemic LNA-miR-29b inhibitor application) results in enhanced elastin fragmentation, increased medial collagen deposition, aortic stiffness and augmented pulse pressure. Conclusions: In conclusion this study identifies miR-29b as a regulator and potential therapeutic target of diabetic aortic stiffening.

Objective— Clinical trials of bone marrow–derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlies their potential efficacy remains unknown. In the present study, we evaluated the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in a murine myocardial infarction model. Methods and Results— We used bioluminescence imaging and high-throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks posttransplantation. Moreover, transcriptomic analysis of BMMCs revealed nonspecific upregulation of various cell regulatory genes, with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography. Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis. Conclusion— Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.

In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α1-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α1-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β1, β2, or both β1- and β2-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β1−/−showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β2−/−developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β1β2−/−) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β1β2−/−compared with the other genotypes, whereas transforming growth factor-β2, a positive mediator of hypertrophy was upregulated in all genotypes except the β1β2−/−. In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β1- and β2-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.

We sought to characterize temporal gene expression changes in the murine angiotensin II (ANG II)-ApoE−/− model of abdominal aortic aneurysm (AAA). Aortic ultrasound measurements were obtained over the 28-day time-course. Harvested suprarenal aortic segments were evaluated with whole genome expression profiling at 7, 14, and 28 days using the Agilent Whole Mouse Genome microarray platform and Statistical Analysis of Microarrays at a false discovery rate of

Background— Network analysis techniques allow a more accurate reflection of underlying systems biology to be realized than traditional unidimensional molecular biology approaches. Using gene coexpression network analysis, we define the gene expression network topology of cardiac hypertrophy and failure and the extent of recapitulation of fetal gene expression programs in failing and hypertrophied adult myocardium. Methods and Results— We assembled all myocardial transcript data in the Gene Expression Omnibus (n=1617). Because hierarchical analysis revealed species had primacy over disease clustering, we focused this analysis on the most complete (murine) dataset (n=478). Using gene coexpression network analysis, we derived functional modules, regulatory mediators, and higher-order topological relationships between genes and identified 50 gene coexpression modules in developing myocardium that were not present in normal adult tissue. We found that known gene expression markers of myocardial adaptation were members of upregulated modules but not hub genes. We identified ZIC2 as a novel transcription factor associated with coexpression modules common to developing and failing myocardium. Of 50 fetal gene coexpression modules, 3 (6%) were reproduced in hypertrophied myocardium and 7 (14%) were reproduced in failing myocardium. One fetal module was common to both failing and hypertrophied myocardium. Conclusions— Network modeling allows systems analysis of cardiovascular development and disease. Although we did not find evidence for a global coordinated program of fetal gene expression in adult myocardial adaptation, our analysis revealed specific gene expression modules active during both development and disease and specific candidates for their regulation.

Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA-26a for additional studies. Inhibition of miRNA-26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA-26a blunted differentiation. As a potential mechanism, we investigated whether miRNA-26a influences TGF-β-pathway signaling. Dual-luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA-26a inhibition, and the opposite effect with miRNA-26a overexpression in transfected human cells. Furthermore, inhibition of miRNA-26a increased gene expression of SMAD-1 and SMAD-4, while overexpression inhibited SMAD-1. MicroRNA-26a was also found to be downregulated in two mouse models of AAA formation (2.5- to 3.8-fold decrease, P < 0.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA-26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF-β pathway signaling. MicroRNA-26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease.