[Show abstract][Hide abstract] ABSTRACT: Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localised MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme.
Journal of Molecular and Cellular Cardiology 06/2014; · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.
[Show abstract][Hide abstract] ABSTRACT: Abdominal aortic aneurysm (AAA) disease is a common, morbid, and highly lethal pathology. Extraordinary efforts have been launched to determine the molecular and pathophysiological characteristics of AAAs. Although surgery is highly effective in preventing death by rupture for larger AAAs, no guidance or preventive therapy is currently available for the >90% of patients whose aneurysms are below the surgical threshold. Predictive animal models of AAA as well as human pathological samples have revealed a complex circuit of AAA formation and progression. The proteolytic destruction of matrix components of the aorta by different proteases has been extensively studied over many years. Recently, a novel class of small noncoding RNAs, called microRNAs, was identified as "fine-tuners" of the translational output of target genes; they act by promoting mRNA degradation. Their therapeutic potential in limiting AAA development appears very intriguing. Further, current studies assessing genetic and heritable associations for AAA disease have provided great insight into its pathogenesis, potentially enabling us to better clinically manage affected patients. Expected final online publication date for the Annual Review of Medicine Volume 65 is January 14, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Annual review of medicine 11/2013; · 9.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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). Whereas physiological mechanical stress helps to maintain vascular structure and function, pathologic or aberrant stress may impair cellular mechano-signaling, and initiate or augment cellular processes which 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.
[Show abstract][Hide abstract] ABSTRACT: Tremendous efforts have been initiated to elucidate the molecular and pathophysiological characteristics of abdominal aortic aneurysm (AAA) disease, which is a significant contributor to morbidity and mortality in the Western world. Recently, a novel class of small noncoding RNAs, called microRNAs, was identified as important transcriptional and posttranscriptional inhibitors of gene expression thought to simultaneously "fine tune" the translational output of multiple target messenger RNAs (mRNAs) by promoting mRNA degradation or inhibiting translation. Several research groups were able to identify the miR-29 family, and miR-29b in particular, as crucial regulators of-not only vascular fibrosis-but also cardiac-, kidney-, liver-, and skin-fibrosis. The current review briefly points out data indicating a causal role for miR-29 in various diseases, while focusing on its potential benefit during AAA initiation and propagation.
Trends in cardiovascular medicine 07/2013; · 4.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 OPEN ACCESS
International Journal of Molecular Sciences 07/2013; 14:14374-14394. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abdominal aortic aneurysms (AAA) are an important source of morbidity and mortality in the U.S. and worldwide. Treatment options are limited, with open surgery or endovascular repair remaining the only curative treatments. Classical cardiovascular medications have generally failed to prevent or significantly alter AAA formation or progression. Therefore, there is a tremendous need for better therapeutic approaches. With increasing knowledge of microRNA (miR) regulation in the context of cardiovascular disease, and with improving technical options permitting alteration of miR-expression levels in vitro and in vivo, we are offered a glimpse into the diagnostic and therapeutic possibilities of using miRs to treat vascular pathobiology. This review focuses on the role of miRs in aneurysmal disease of the abdominal aorta, summarizing recent publications regarding this topic, and outlining known effects of relevant miRs in AAA formation, including miR-21 and miR-29b. Despite there being only limited studies available, several other miRs also display clear potential for alteration of the disease process including miR-26a, the miR-17-92-cluster, miRs-221/222, miR-133 and miR-146a. While studies have shown that miRs can regulate the activity and interplay of vascular inflammatory cells, endothelial cells, smooth muscle cells and fibroblasts, all key elements leading to AAA formation, much work remains to be done.
Current Vascular Pharmacology 05/2013; · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE: Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear. METHODS AND RESULTS: Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow-derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly attributable in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model. CONCLUSIONS: These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.
Arteriosclerosis Thrombosis and Vascular Biology 11/2012; · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Two direct measurements of peripheral insulin sensitivity are the M value derived from the euglycemic, hyperinsulinemic clamp (EC) and the steady-state plasma glucose (SSPG) concentration derived from the insulin suppression test (IST). Prior work suggests that these measures are highly correlated, but the agreement between them is unknown. To determine the agreement between SSPG and M and to develop transformation equations to convert SSPG to M and vice versa, we directly compared these two measurements in the same individuals. METHODS: A total of 15 nondiabetic subjects (9 women and 6 men) underwent both an EC and a modified version of the IST within a median interval of 5days. We performed standard correlation metrics of the two measures and developed transformation regression equations for the two measures. RESULTS: The mean±SD age of the subjects was 57±7years and body mass index, 27.7±3.9kg/m(2). The median (interquartile range) SSPG concentration was 6.7 (5.1, 9.8) mmol/L and M value, 49.6 (28.9, 64.2) μmol/min/kg-LBM. There was a highly significant correlation between SSPG and M (r=-0.87, P <0.001). The relationship was best fit by regression models with exponential/logarithmic functions (R(2)=0.85). Bland-Altman plots demonstrated an excellent agreement between these measures of insulin action. CONCLUSION: The SSPG and M are highly related measures of insulin sensitivity and the results provide the means to directly compare the two measurements.
Metabolism: clinical and experimental 11/2012; · 3.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Many coronary heart disease (CHD) events occur in individuals classified as intermediate risk by commonly used assessment tools. Over half the individuals presenting with a severe cardiac event, such as myocardial infarction (MI), have at most one risk factor as included in the widely used Framingham risk assessment. Individuals classified as intermediate risk, who are actually at high risk, may not receive guideline recommended treatments. A clinically useful method for accurately predicting 5-year CHD risk among intermediate risk patients remains an unmet medical need.
This study sought to develop a CHD Risk Assessment (CHDRA) model that improves 5-year risk stratification among intermediate risk individuals.
Assay panels for biomarkers associated with atherosclerosis biology (inflammation, angiogenesis, apoptosis, chemotaxis, etc.) were optimized for measuring baseline serum samples from 1084 initially CHD-free Marshfield Clinic Personalized Medicine Research Project (PMRP) individuals. A multivariable Cox regression model was fit using the most powerful risk predictors within the clinical and protein variables identified by repeated cross-validation. The resulting CHDRA algorithm was validated in a Multiple-Ethnic Study of Atherosclerosis (MESA) case-cohort sample.
A CHDRA algorithm of age, sex, diabetes, and family history of MI, combined with serum levels of seven biomarkers (CTACK, Eotaxin, Fas Ligand, HGF, IL-16, MCP-3, and sFas) yielded a clinical net reclassification index of 42.7% (p < 0.001) for MESA patients with a recalibrated Framingham 5-year intermediate risk level. Across all patients, the model predicted acute coronary events (hazard ratio = 2.17, p < 0.001), and remained an independent predictor after Framingham risk factor adjustments.
These include the slightly different event definition with the MESA samples and inability to include PMRP fatal CHD events.
A novel risk score of serum protein levels plus clinical risk factors, developed and validated in independent cohorts, demonstrated clinical utility for assessing the true risk of CHD events in intermediate risk patients. Improved accuracy in cardiovascular risk classification could lead to improved preventive care and fewer deaths.
Current Medical Research and Opinion 10/2012; 28(11):1819. · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apelin is an endogenous peptide hormone recently implicated in glucose homeostasis. However, whether apelin affects glucose uptake in myocardial tissue remains undetermined. In this study, we utilized in vivo, ex vivo and in vitro methods to study apelin's effect on myocardial glucose uptake. Pyroglutamated apelin-13 (2mg/kg/day) was administered to C57BL6/J mice for 7 days. In vivo myocardial glucose uptake was measured by FDG-PET scanning, and GLUT4 translocation was assessed by immunofluorescence imaging. For in vitro studies, differentiated H9C2 cardiomyoblasts were exposed to pyroglutamated apelin-13 (100nM) for 2h. To test their involvement in apelin-stimulated myocardial glucose uptake, the energy sensing protein kinase AMPK were inhibited by pharmacologic inhibition (compound C) and RNA interference. IRS-1 phosphorylation was assessed by western blotting using an antibody directed against IRS-1 Ser-789-phosphorylated form. We found that apelin increased myocardial glucose uptake and GLUT4 membrane translocation in C57BL6/J mice. Apelin was also sufficient to increase glucose uptake in H9C2 cells. Apelin-mediated glucose uptake was significantly decreased by AMPK inhibition. Finally, apelin increased IRS-1 Ser-789 phosphorylation in an AMPK-dependent manner. The results of our study demonstrated that apelin increases myocardial glucose uptake through a pathway involving AMPK. Apelin also facilitates IRS-1 Ser-789 phosphorylation, suggesting a novel mechanism for its effects on glucose uptake.
[Show abstract][Hide abstract] ABSTRACT: Differentiated vascular smooth muscle cells (SMCs) retain the capacity to modify their phenotype in response to inflammation or injury. This phenotypic switching is a crucial component of vascular disease, and is partly dependent on epigenetic regulation. An appreciation has been building in the literature for the essential role chromatin remodelling plays both in SMC lineage determination and in influencing changes in SMC behaviour and state. This process includes numerous chromatin regulatory elements and pathways such as histone acetyltransferases, deacetylases, and methyltransferases and other factors that act at SMC-specific marker sites to silence or permit access to the cellular transcriptional machinery and on other key regulatory elements such as myocardin and Kruppel-like factor 4 (KLF4). Various stimuli known to alter the SMC phenotype, such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), oxidized phospholipids, and retinoic acid, appear to act in part through effects upon SMC chromatin structure. In recent years, specific covalent histone modifications that appear to establish SMC determinacy have been identified, while others alter the differentiation state. In this article, we review the mechanisms of chromatin remodelling as it applies to the SMC phenotype.
Cardiovascular research 02/2012; 95(2):147-55. · 5.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Identification and treatment of abdominal aortic aneurysm (AAA) remains among the most prominent challenges in vascular medicine. MicroRNAs are crucial regulators of cardiovascular pathology and represent possible targets for the inhibition of AAA expansion. We identified microRNA-21 (miR-21) as a key modulator of proliferation and apoptosis of vascular wall smooth muscle cells during development of AAA in two established murine models. In both models (AAA induced by porcine pancreatic elastase or infusion of angiotensin II), miR-21 expression increased as AAA developed. Lentiviral overexpression of miR-21 induced cell proliferation and decreased apoptosis in the aortic wall, with protective effects on aneurysm expansion. miR-21 overexpression substantially decreased expression of the phosphatase and tensin homolog (PTEN) protein, leading to increased phosphorylation and activation of AKT, a component of a pro-proliferative and antiapoptotic pathway. Systemic injection of a locked nucleic acid-modified antagomir targeting miR-21 diminished the pro-proliferative impact of down-regulated PTEN, leading to a marked increase in the size of AAA. Similar results were seen in mice with AAA augmented by nicotine and in human aortic tissue samples from patients undergoing surgical repair of AAA (with more pronounced effects observed in smokers). Modulation of miR-21 expression shows potential as a new therapeutic option to limit AAA expansion and vascular disease progression.
Science translational medicine 02/2012; 4(122):122ra22. · 10.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRs) regulate gene expression at the posttranscriptional level and play crucial roles in vascular integrity. As such, they may have a role in modifying abdominal aortic aneurysm (AAA) expansion, the pathophysiological mechanisms of which remain incompletely explored. Here, we investigate the role of miRs in 2 murine models of experimental AAA: the porcine pancreatic elastase (PPE) infusion model in C57BL/6 mice and the AngII infusion model in Apoe-/- mice. AAA development was accompanied by decreased aortic expression of miR-29b, along with increased expression of known miR-29b targets, Col1a1, Col3a1, Col5a1, and Eln, in both models. In vivo administration of locked nucleic acid anti-miR-29b greatly increased collagen expression, leading to an early fibrotic response in the abdominal aortic wall and resulting in a significant reduction in AAA progression over time in both models. In contrast, overexpression of miR-29b using a lentiviral vector led to augmented AAA expansion and significant increase of aortic rupture rate. Cell culture studies identified aortic fibroblasts as the likely vascular cell type mediating the profibrotic effects of miR-29b modulation. A similar pattern of reduced miR-29b expression and increased target gene expression was observed in human AAA tissue samples compared with that in organ donor controls. These data suggest that therapeutic manipulation of miR-29b and its target genes holds promise for limiting AAA disease progression and protecting from rupture.
The Journal of clinical investigation 02/2012; 122(2):497-506. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous clinical studies in pulmonary arterial hypertension (PAH) have concentrated predominantly on distal pulmonary vascular resistance, its contribution to the disease process, and response to therapy. However, it is well known that biomechanical factors such as shear stress have an impact on endothelial health and dysfunction in other parts of the vasculature. This study tested the hypothesis that wall shear stress is reduced in the proximal pulmonary arteries of PAH patients with the belief that reduced shear stress may contribute to pulmonary endothelial cell dysfunction and as a result, PAH progression. A combined MRI and computational fluid dynamics (CFD) approach was used to construct subject-specific pulmonary artery models and quantify flow features and wall shear stress (WSS) in five PAH patients with moderate-to-severe disease and five age- and sex-matched controls. Three-dimensional model reconstruction showed PAH patients have significantly larger main, right, and left pulmonary artery diameters (3.5 ± 0.4 vs. 2.7 ± 0.1 cm, P = 0.01; 2.5 ± 0.4 vs. 1.9 ± 0.2 cm, P = 0.04; and 2.6 ± 0.4 vs. 2.0 ± 0.2 cm, P = 0.01, respectively), and lower cardiac output (3.7 ± 1.2 vs. 5.8 ± 0.6 L/min, P = 0.02.). CFD showed significantly lower time-averaged central pulmonary artery WSS in PAH patients compared to controls (4.3 ± 2.8 vs. 20.5 ± 4.0 dynes/cm(2), P = 0.0004). Distal WSS was not significantly different. A novel method of measuring WSS was utilized to demonstrate for the first time that WSS is altered in some patients with PAH. Using computational modeling in patient-specific models, WSS was found to be significantly lower in the proximal pulmonary arteries of PAH patients compared to controls. Reduced WSS in proximal pulmonary arteries may play a role in the pathogenesis and progression of PAH. This data may serve as a basis for future in vitro studies of, for example, effects of WSS on gene expression.
[Show abstract][Hide abstract] ABSTRACT: In our previous transcriptional profiling of a murine model, we have identified a remarkably small number of specific pathways with altered expression in lymphedema. In this investigation, we utilized microarray-based transcriptomics of human skin for an unbiased a priori prospective candidate identification, with subsequent validation of these candidates through direct serum assay. The resulting multi-analyte biomarker panel sensitively should sensitively discriminate human lymphedema subjects from normal individuals.
We enrolled 63 lymphedema subjects and 27 normals in our attempt to discover protein analytes that can distinguish diseased individuals from controls. To minimize technical and biologically irrelevant variation, we first identified potential candidates by performing transcriptional microarray analysis on paired diseased and normal skin specimens sampled from the same individuals. We focused our attention on genes with corresponding protein products that are secreted and took these candidates forward to a protein multiplex assay applied to diseased and normal subjects. We developed a logistic regression-based model on an eventual group of six proteins and validated our system on a separate cohort of study subjects. The area under the receiver operating characteristic curve was calculated to be 0.87 (95% CI : 0.75 to 0.97).
WE HAVE DEVELOPED AN ACCURATE BIOASSAY UTILIZING PROTEINS REPRESENTING FOUR CENTRAL PATHOGENETIC MODALITIES OF THE DISEASE: lymphangiogenesis, inflammation, fibrosis, and lipid metabolism, suggesting that these proteins are directly related to the pathogenesis of the tissue pathology in lymphatic vascular insufficiency. Further studies are warranted to determine whether this newly-identified biomarker panel will possess utility as an instrument for in vitro diagnosis of early and latent disease; the ultimate applicability to risk stratification, quantitation of disease burden, and response to therapy can easily be envisioned.
PLoS ONE 01/2012; 7(12):e52021. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic inflammation is considered to play a role in the development of cardiovascular disease. Various (n-3) fatty acids (FA) have been reported to have antiinflammatory effects, but there is a lack of consensus in this area, particularly in regard to optimal source(s) and dose(s). This study aimed to determine the effects of high and low doses of (n-3) FA from plant and marine sources on plasma inflammatory marker concentrations. One-hundred adults with metabolic syndrome were randomly assigned to a low or high dose of plant- (2.2 or 6.6 g/d α-linolenic acid) or marine- (1.2 or 3.6 g/d EPA and DHA) derived (n-3) FA or placebo for 8 wk, using a parallel arm design (n = 20/arm). Fasting blood samples collected at 0, 4, and 8 wk were analyzed for concentrations of monocyte chemotactic protein-1 (MCP-1), IL-6, and soluble intercellular adhesion molecule-1 (sICAM-1) and for cardiovascular risk factors. Baseline concentrations across all 5 groups combined were (mean ± SD) 103 ± 32 ng/L for MCP-1, 1.06 ± 0.56 ng/L for IL-6, and 0.197 ± 0.041 ng/L for sICAM-1. There were no significant differences in 8-wk changes in plasma inflammatory marker concentrations among the 5 groups. Plasma TG and blood pressure decreased significantly more and the LDL cholesterol concentration increased more in the high-dose fish oil group compared to the 8-wk changes in some of the other 4 groups (P ≤ 0.04). In conclusion, no beneficial effects were detected for any of the 3 inflammatory markers investigated in response to (n-3) FA in adults with metabolic syndrome regardless of dose or source.
Journal of Nutrition 12/2011; 141(12):2166-71. · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study investigated the spatial and temporal remodeling of blood vessel wall microarchitecture and cellular morphology during abdominal aortic aneurysm (AAA) development using immunofluorescent array tomography (IAT), a high-resolution three-dimensional (3D) microscopy technology, in the murine model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7, and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified volume fractions (VF) of elastin, smooth muscle cell (SMC) actin, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, non-adventitial wall thickness, and nuclei amount. The 3D renderings depicted elastin and collagen type I degradation and SMC morphological changes. Elastin VF decreased 37.5% (p<0.01), thickness decreased 48.9%, and fragmentation increased 449.7% (p<0.001) over 28 days. SMC actin VF decreased 78.3% (p<0.001) from days 0 to 7 and increased 139.7% (p<0.05) from days 7 to 28. Non-adventitial wall thickness increased 61.1%, medial nuclei amount increased 159.1% (p<0.01), and adventitial collagen type I VF decreased 64.1% (p<0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the dynamics of vascular remodeling during AAA development.
Journal of Histochemistry and Cytochemistry 12/2011; 60(2):97-109. · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Marfan syndrome (MFS) is a systemic connective tissue disorder notable for the development of aortic root aneurysms and the subsequent life-threatening complications of aortic dissection and rupture. Underlying fibrillin-1 gene mutations cause increased transforming growth factor-β (TGF-β) signaling. Although TGF-β blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF-β causes aneurysms remain ill-defined.
We investigated the role of microRNA-29b (miR-29b) in aneurysm formation in MFS.
Using quantitative polymerase chain reaction, we discovered that miR-29b, a microRNA regulating apoptosis and extracellular matrix synthesis/deposition genes, is increased in the ascending aorta of Marfan (Fbn1(C1039G/+)) mice. Increased apoptosis, assessed by increased cleaved caspase-3 and caspase-9, enhanced caspase-3 activity, and decreased levels of the antiapoptotic proteins, Mcl-1 and Bcl-2, were found in the Fbn1(C1039G/+) aorta. Histological evidence of decreased and fragmented elastin was observed exclusively in the Fbn1(C1039G/+) ascending aorta in association with repressed elastin mRNA and increased matrix metalloproteinase-2 expression and activity, both targets of miR-29b. Evidence of decreased activation of nuclear factor κB, a repressor of miR-29b, and a factor suppressed by TGF-β, was also observed in Fbn1(C1039G/+) aorta. Furthermore, administration of a nuclear factor κB inhibitor increased miR-29b levels, whereas TGF-β blockade or losartan effectively decreased miR-29b levels in Fbn1(C1039G/+) mice. Finally, miR-29b blockade by locked nucleic acid antisense oligonucleotides prevented early aneurysm development, aortic wall apoptosis, and extracellular matrix deficiencies.
We identify increased miR-29b expression as key to the pathogenesis of early aneurysm development in MFS by regulating aortic wall apoptosis and extracellular matrix abnormalities.
Circulation Research 11/2011; 110(2):312-24. · 11.86 Impact Factor